Polymer conjugates of glp-1

ABSTRACT

Conjugates of a GLP-1 moiety may be covalently attached to one or more water-soluble polymers. For instance, a GLP-1 polymer conjugate may include a GLP-1 moiety releasably attached at its N-terminus to a water-soluble polymer. The GLP-1 polymer conjugate may include a GLP-1 moiety covalently attached to a water-soluble polymer, wherein the GLP-1 moiety possesses an N-methyl substituent.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/086,687, filed Nov. 18, 2009, which is a 35 U.S.C. §371 applicationof International Application No. PCT/US2006/048181, filed Dec. 18, 2006,which claims priority to U.S. Application No. 60/751,121, filed Dec. 16,2005, and to U.S. Application No. 60/751,082, filed Dec. 16, 2005, allof which are hereby incorporated by reference in their entireties. Thepresent application expressly incorporates by reference herein theentire disclosure of U.S. Provisional Patent Application Ser. No.60/691,516, filed Jun. 16, 2005.

FIELD OF THE INVENTION

The present invention relates generally to pharmaceuticals. Forinstance, the present invention relates to conjugates comprising a GLP-1(glucagon-like peptide-1) moiety covalently attached to one or morewater-soluble polymers. Among other things, the invention additionallyrelates to methods for synthesizing GLP-1 polymer conjugates,compositions comprising such conjugates, and methods for treatingpatients by administering GLP-1 conjugates.

BACKGROUND OF THE INVENTION

Glucagon-like-peptide-1 (referred to hereinafter as GLP-1) is aproglucagon-derived peptide secreted from intestinal L-cells in responseto nutrient ingestion (Drucker, D J: The Glucagon-Like Peptides.Diabetes 47:159-169, 1998). GLP-1 acts as an incretin to stimulate therelease of insulin from pancreatic beta cells in conjunction withcarbohydrates that are absorbed from the gut. GLP-1 also exerts actionsindependent of islet hormone secretion, including inhibition of bothgastric emptying and food intake and stimulation of β-cellproliferation.

Significantly, GLP-1 possesses the ability to rapidly lower glucoselevels in both normal and diabetic subjects (Gutniak, M., et al., N EnglJ Med 326:1316-1322, 1992; Nauck M A, et al., J Clin Invest 91:301-307,1993). In a six-week study in humans, continuous subcutaneous infusionof native GLP-1 significantly decreased blood glucose and HbA_(1c) inpatients with type 2 diabetes (Zander M, et al., Lancet 359:824-830,2002). Based on results such as these, there has been considerableinterest in developing GLP-1 based pharmaceutical agents for thetreatment of type-2 diabetes and the like.

Although native GLP-1 effectively lowers blood glucose followingadministration, its usefulness as a therapeutic agent is severelyhampered due to the fact that native GLP-1 (amino acids 1-37) is poorlyactive, and its two naturally-occurring truncated versions, GLP-1 (7-37)and GLP-1 (7-36)NH₂, have extremely short in vivo half-lives. The shortin vivo half-life of GLP-1 is due primarily to NH₂-terminal cleavage andinactivation by the enzyme, dipeptidyl peptidase, DPP-IV (Kieffer T J etal., Endocrinology 136:3585-3596, 1995).

Various approaches have been explored to attempt to circumvent the rapidin vivo cleavage of GLP-1 by DPP-IV. For example, GLP-1 analogs havingone or more amino acid substitutions aimed at reducing the affinity ofGLP-1 for DPP-IV to attenuate its cleavage have been prepared (Drucker DJ, Curr Pharm Des 7:1399-1412, 2001; Drucker D J, Gastroenterology122:531-544, 2002). Similarly, the naturally-occurring lizard peptide,exendin-4, has been found to be a potent GLP-1R agonist that exhibitsreduced DPP-IV mediated cleavage and possesses a longer duration ofaction than GLP-1 (Young, A A, et al., Diabetes 48:1026-1034, 1999;Edwards C M, et al., Am J Physiol Endicrinol Metab 281:E155-161, 2001).GLP-1 derivatives having a short covalent chemical linker designed tointeract with a specific cysteine residue in albumin followingadministration have also been developed (Kim, J G, et al., Diabetes 52,751, 2003), as have GLP-1 analogs having several modifications includinga modified N-terminus, an octanoic acid acylated at lysine-34, and asubstitution of arginine at position 26 (Chou, J., et al., J. Pharm Sci,86 (7), 768-773, 2000). Additionally, stable covalent attachment ofpolyethylene glycol to GLP-1 has been described (See, e.g., WO2004/093823).

While these and other various approaches appear to describe GLP-1compounds having improved therapeutic properties when compared to nativeGLP-1, there still exists a need for improved GLP-1 compounds capable ofproviding additional therapeutic advantages over existing GLP-1 basedtherapeutic agents. Thus, there remains a need in the art to provideadditional advantageous GLP-1 moiety-polymer conjugates. Among otherthings, one or more embodiments of the present invention are thereforedirected to such conjugates as well as compositions comprising theconjugates and related methods as described herein.

SUMMARY OF THE INVENTION

Accordingly, the present invention provides GLP-1 conjugates. Otherfeatures and advantages of the present invention will be set forth inthe description of invention that follows, and in part will be apparentfrom the description or may be learned by practice of the invention. Theinvention will be realized and attained by the compositions and methodsparticularly pointed out in the written description and claims hereof.

One aspect of the present invention is directed to a GLP-1 polymerconjugate comprising a GLP-1 moiety releasably attached at itsN-terminus to a water-soluble polymer.

Another aspect of the present invention is directed to a pharmaceuticalcomposition comprising at least an effective amount of a mixture ofpositional isomers of GLP-1 mono-polymer conjugates having a singlewater-soluble polymer releasably attached to a GLP-1 moiety, wherein oneof the mono-polymer conjugates possesses the water-soluble polymercovalently attached to the N-terminus of the GLP-1 moiety.

Yet another aspect of the present invention is directed to apharmaceutical composition comprising at least an effective amount of amixture of GLP-1 polymer conjugates, wherein at least one GLP-1 polymerconjugate possesses a water-soluble polymer releasably attached to aGLP-1 moiety at its N-terminus.

Still another aspect of the present invention is directed to a methodfor preparing a GLP-1 polymer conjugate, the method comprisingcontacting a GLP-1 moiety with a water-soluble polymer reagentcomprising an amino-reactive functional group suitable to form ahydrolyzable linkage, under conjugation conditions effective to promotereaction of the N-terminal amino group of the GLP-1 moiety with theamino-reactive functional group of the water-soluble polymer reagent, tothereby form a GLP-1 polymer conjugate comprising the GLP-1 moietyreleasably attached at its N-terminus to the water-soluble polymer.

In another aspect the present invention is directed to a method fortreating a condition in a mammalian subject responsive to treatment withGLP-1, the method comprising administering to the subject, a GLP-1polymer conjugate comprising a GLP-1 moiety releasably attached to awater-soluble polymer, wherein the conjugate lacks bioactivity prior tothe administering, and whereby, as a result of the administering, theGLP-1 moiety is released from the conjugate and is effective to resultin a reduction in blood glucose level in the subject over a period oftime prolonged over that observed for native GLP-1.

In yet another aspect the present invention is directed to a GLP-1polymer conjugate comprising a GLP-1 moiety covalently attached to awater-soluble polymer, wherein the GLP-1 moiety possesses an N-methylsubstituent.

In still another aspect the present invention is directed to a GLP-1polymer conjugate comprising a GLP-1 moiety covalently attached at apolymer attachment site to a water-soluble polymer, wherein the GLP-1moiety is glycosylated at a site separate from the polymer attachmentsite.

In another aspect the present invention is directed to a composition,comprising a glycosylated and pegylated GLP-1 conjugate made by stepwisesolid phase peptide synthesis involving contacting a growing peptidechain with protected amino acids in a stepwise manner, wherein at leastone of the protected amino acids is glycosylated, followed bypegylation, and wherein the composition has a purity of at least about95% of a single species of the glycosylated and pegylated GLP-1conjugate.

In yet another aspect the present invention is directed to a method ofmaking a glycosylated GLP-1 polymer conjugate. The method includescontacting a growing peptide chain with protected amino acids in astepwise manner, wherein at least one of the protected amino acids isglycosylated prior to contacting, under conditions effective to form aglycosylated GLP-1. The method also includes contacting the glycosylatedGLP-1 with a water-soluble polymer reagent under conjugation conditionseffective to form the glycosylated GLP-1 polymer conjugate comprising aglycosylated GLP-1 moiety attached to the water-soluble polymer.

In still another aspect the present invention is directed to a GLP-1polymer conjugate having the structure:

wherein:POLY¹ is a first water-soluble polymer;POLY² is a second water-soluble polymer;X¹ and X² are each independently a spacer moiety having an atom lengthof from about 1 to about 18 atoms;

is an aromatic-containing moiety bearing an ionizable hydrogen atom,H_(α);R¹ is H or a lower alkyl;R² is H or a lower alkyl;

Y¹ is O or S; Y² is O or S; and

—NH-GLP-1 is a GLP-1 moiety, wherein the —NH— of —NH-GLP-1 represents anamino group of the GLP-1 moiety.

In yet another aspect the present invention is directed to a GLP-1polymer conjugate having the structure:

wherein:POLY¹ is a first water-soluble polymer;POLY² is a second water-soluble polymer;X¹ and X² are each independently a spacer moiety having an atom lengthof from about 1 to about 18 atoms;Ar¹ is a first aromatic moiety;Ar² is a second aromatic moiety;Hα is an ionizable hydrogen atom;R¹ is H or a lower alkyl;R² is H or a lower alkyl;

Y¹ is O or S; Y² is O or S; and

—NH-GLP-1 is a GLP-1 moiety, wherein the —NH— of —NH-GLP-1 represents anamino group of the GLP-1 moiety.

In another aspect the present invention is directed to a GLP-1 polymerconjugate having the structure:

wherein:POLY¹ is a first water-soluble polymer;POLY² is a second water-soluble polymer;X¹ and X² are each independently a spacer moiety having an atom lengthof from about 1 to about 18 atoms;Ar¹ is a first aromatic moiety;Ar² is a second aromatic moiety;Hα is an ionizable hydrogen atom;R¹ is H or a lower alkyl;R² is H or a lower alkyl;

Y¹ is O or S; Y² is O or S; and

—NH-GLP-1 is a GLP-1 moiety, wherein the —NH— of —NH-GLP-1 represents anamino group of the GLP-1 moiety.

In still another aspect the present invention is directed to a GLP-1polymer conjugate having the structure:

wherein:POLY¹ is a first water-soluble polymer;POLY² is a second water-soluble polymer;X¹, X², and X³ are each independently a spacer moiety having an atomlength of from about 1 to about 18 atoms;Ar¹ is a first aromatic moiety;Ar² is a second aromatic moiety;Hα is an ionizable hydrogen atom;R¹ is H or a lower alkyl;R² is H or a lower alkyl;

Y¹ is O or S; Y² is O or S; and

—NH-GLP-1 is a GLP-1 moiety, wherein the —NH— of —NH-GLP-1 represents anamino group of the GLP-1 moiety.

Accordingly, in one aspect, the invention provides extended deliverycompositions comprising a GLP-1 moiety. The conjugates and compositionsof the invention typically possess extended release properties in vitroand in vivo. In vivo animal model data presented herein suggests thatthe GLP-1 conjugates of the invention can possess a longer circulatinghalf-life in the bloodstream than native GLP-1, thereby overcoming someof the drawbacks associated with therapeutic administration of nativeGLP-1 due to its rapid clearance.

Thus, the conjugates and compositions described herein may facilitate adecreased frequency of dosing compared to native GLP-1.

For example, in one or more embodiments, a GLP-1 polymer conjugate ofthe invention is releasable, e.g., is effective to release the GLP-1moiety, e.g., by a degradative process such as hydrolysis. Suchreleasable conjugates will comprise at least one degradable linkage,such as a hydrolyzable linkage, between the GLP-1 moiety and thewater-soluble polymer.

In one or more embodiments, the water-soluble polymer is selected fromthe group consisting of poly(alkylene glycols), poly(oxyethylatedpolyols), poly(olefinic alcohols), poly(vinylpyrrolidone),poly(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate),poly(saccharides), poly(α-hydroxy acid), poly(vinyl alcohol),polyphosphazene, polyoxazoline, poly(N-acryloylmorpholine), andcopolymers and terpolymers thereof.

In some preferred embodiments, the water-soluble polymer is apolyethylene glycol.

A GLP-1 polymer conjugate of the invention may additionally comprise atleast one additional water-soluble polymer such as polyethylene glycolreleasably attached to a number of sites on the GLP-1 moiety selectedfrom one and two. Such exemplary sites on the GLP-1 moiety include Lys26and Lys34, among others.

In one or more embodiments, the GLP-1 polymer conjugate possesses thewater-soluble polymer, e.g., polyethylene glycol, releasably attached toa single site of the GLP-1 moiety.

In yet one or more embodiments, the water-soluble polymer is releasablyattached to the GLP-1 moiety via a linker comprising a hydrolyzablelinkage selected from carbamate, carboxylate ester, phosphate ester,anhydride, acetal, ketal, acyloxyalkyl ether, imine, orthoester,thioester, thiolester, and carbonate. Particularly preferred linkagesinclude carbamate, carboxylate ester, and carbonate.

In yet one or more additional embodiments, a GLP-1 conjugate of theinvention comprises a GLP-1 moiety releasably attached, e.g., via ahydrolyzable linkage, to a water-soluble polymer such as PEG, such thatupon hydrolysis either in vitro or in vivo, the unconjugated GLP-1moiety is released. Due to the hydrolyzable nature of such conjugates, aGLP-1 conjugate of the invention may or may not be bioactive.

Thus, in one or more embodiments, a GLP-1 polymer conjugate is providedwhere the conjugate essentially lacks bioactivity, yet is stilleffective, when administered in vivo to a mammalian subject in needthereof, to provide a blood glucose-lowering effect. Such blood glucoselowering is achieved by release of the GLP-1 moiety subsequent toadministration.

In one or more embodiments, the water-soluble polymer has a molecularweight ranging from about 500 daltons to about 80,000 daltons, such asfrom about 1000 daltons to about 40,000 daltons.

In yet one or more additional embodiments, the water-soluble polymer hasa structure selected from linear, branched, forked, and multi-armed.

In yet one or more further embodiments, the GLP-1 moiety isglycosylated.

In a preferred embodiment, the glycosylated GLP-1 possesses a mono-,di-, or trisaccharide covalently attached to one or more of its aminoacid sites. For example, the mono-, di-, or trisaccharide may becovalently attached to one or more sites of the GLP-1 moiety, whereinthe one or more sites each comprise one of Asp, Asn, Ser, and Thr thatis naturally occurring or substituted at one or more sites selected fromthe N-terminus (His7), Ala8, Glu9, Thr11, Thr13, Ser14, Ser17, Ser18,Glu21, Gly22, Gln23, Lys26, and Lys34.

In yet one or more additional embodiments, a GLP-1 polymer conjugatepossesses a mono-, di-, or trisaccharide covalently attached to one ofAsp, Asn, Ser, and Thr that is substituted at the Ala8 site of the GLP-1moiety. Illustrative saccharides include glucose, mannose, xylose,lactose, maltose, melibiose, and maltotriose. Such GLP-1 polymerconjugates may further comprise a mono-, di- or trisaccharide covalentlyattached at one or both of positions Glu21 and Gln23.

In yet one or more further embodiments, the GLP-1 polymer conjugatepossesses an N-methyl substituent at any one or more of positions 7-His,8-Ala, and 9-glutamic acid of the GLP-1 moiety.

In one or more embodiments, a GLP-1 polymer conjugate possesses thefollowing structure:

where POLY is a water-soluble polymer, L_(D) is a degradable linkage,e.g., a hydrolyzable linkage, and k corresponds to the number ofreactive sites on GLP-1 to which an independent polymer segment(POLY-L_(D)) is covalently attached. GLP-1 is a GLP-1 moiety. Each ofthe polymer segments is independently selected, although typically, eachof the polymer segments covalently attached to the GLP-1 moiety is thesame. Typically, k ranges from about 1 to about 4, that is to say, isselected from the group consisting of 1, 2, 3, and 4. Often, k is 1, 2,or 3. In some particular embodiments, k is 1. In yet one or morespecific embodiments, L_(D) is —O—C(O)—NH—.

Generally, the L_(D) in the above structure possesses a length such asfrom about 1 to about 20 atoms, from about 2 to about 15 atoms, or fromabout 3 to about 10 atoms. That is to say, typically, L_(D) has anoverall atom length selected from the group consisting of 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20.

In one or more particular embodiments, a GLP-1 polymer conjugatepossesses the following structure:

where

mPEG is CH₃O—(CH₂CH₂O)_(n)CH₂CH₂—,

n ranges from 10 to 1800,

p is an integer ranging from 1 to 8,

R₁ is H or lower alkyl,

R₂ is H or lower alkyl,

Ar is an aromatic hydrocarbon, such as a bicyclic or tricyclic aromatic,

X₁ and X₂ are each independently a spacer moiety having an atom lengthof from about 1 to about 18 atoms, and

—NH-GLP-1 is a GLP-1 moiety, wherein the —NH— of —NH-GLP-1 represents anamino group of the GLP-1 moiety.

In references to the preceding structure, in one or more specificembodiments, p is 1 and R₁ and R₂ are both H. In yet one or morealternative embodiments, X₁ and X₂ each comprise at least one amidebond. In yet one or more additional embodiments, X₁ and X₂ are the same.

In reference to the preceding structure, X₁ and X₂, may, in certainembodiments, each independently possess a structure selected from—NH—C(O)—CH₂—O—, —NH—C(O)—(CH₂)_(q)—O—, —NH—C(O)—(CH₂)_(q)—C(O)—NH—,—NH—C(O)—(CH₂)_(q)—, and —C(O)—NH—, where q is selected from 2, 3, 4,and 5. In yet one or more further embodiments, Ar is selected frompentalene, indene, naphthalene, indacene, acenaphthylene, and fluorene.

Illustrative GLP-1 polymer conjugates of the invention include thefollowing releasable conjugates:

In yet one or more additional embodiments, a releasable GLP-1 polymerconjugate of the invention possesses the following generalizedstructure:

where L_(D1) is either —O— or —NH—C(O)—, Ar₁ is an aromatic group,—NH-GLP-1 is a GLP-1 moiety, wherein the —NH— of —NH-GLP-1 represents anamino group of the GLP-1 moiety, and k is selected from 1, 2, and 3.Preferably, Ar₁ is selected from ortho, meta, and para-substitutedphenyl. Illustrative conjugates of this type include:

In yet one or more additional embodiments, a GLP-1 polymer conjugate ofthe invention is characterized by the following structure:

where n ranges from about 10 to about 1800.

In one or more related embodiments, the GLP-1 moiety possesses anN-methyl substituent at any one or more of positions 7-His, 8-Ala, and9-Glu.

Also forming part of the invention is a pharmaceutical compositioncomprising any one or more of the herein described GLP-1 polymerconjugates, e.g., in combination with a pharmaceutically acceptableexcipient.

In one or more embodiments, the pharmaceutical composition comprises, inaddition to the GLP-1 conjugate, insulin and/or basal insulin.

In yet one or more alternative embodiments, the pharmaceuticalcomposition comprises, in addition to the GLP-1 conjugate, GLP-1.

Preferred compositions include those suitable for parenteral orpulmonary administration.

In one or more embodiments, a pharmaceutical composition of theinvention is in dry powder form.

In one of more particular embodiments, the composition as describedabove comprises a GLP-1 polymer conjugate having the water-solublepolymer covalently attached, either releasably or stably, to the GLP-1moiety at the side chain of His7 at the imidazole nitrogen.

As noted above, in certain aspects, the invention provides a method forpreparing a GLP-1 polymer conjugate. In certain embodiments, the methodfurther comprises isolating the GLP-1 polymer conjugate.

In yet one or more additional embodiments, the method further comprisespurifying the GLP-1 polymer conjugate. For example, purifying can be bychromatography or membrane separation.

In one or more embodiments of the method, the GLP-1 moiety employed inthe contacting step comprises protected 8-amino lysines.

In yet a related embodiment, the method may further comprise subsequentto the contacting, deprotecting the protected ε-amino lysines of theGLP-1 polymer conjugate.

In yet another embodiment, provided herein is an aerosolized compositioncomprising a GLP-1 polymer conjugate.

As noted above, in certain aspects, the invention provides a method fordelivery of a GLP-1 polymer conjugate to a mammalian subject in needthereof. In one or more related embodiments of this aspect of theinvention, the administering is by a route selected from subcutaneousand inhalation.

In yet one or more further embodiments, the administering is byinhalation and the GLP-1 polymer conjugate is in aerosolized form.

Yet in another aspect, the invention provides a method for delivery of aGLP-1 moiety to a mammalian subject in need thereof, where the methodcomprises pulmonarily administering to the subject a therapeuticallyeffective amount of a GLP-1 polymer conjugate.

In yet another aspect, provided herein is a method for delivering aGLP-1 moiety to a mammalian subject. The method comprises the steps ofaerosolizing a pharmaceutical composition comprising a GLP-1 polymerconjugate to form an aerosolized composition, and administering theaerosolized composition to the lungs of the subject by inhalation.

In yet another aspect, provided is a method for treating a condition ina mammalian subject responsive to treatment with GLP-1. The methodcomprises administering to the subject, a GLP-1 polymer conjugatecomprising a GLP-1 moiety releasably attached to a water-solublepolymer, wherein the conjugate lacks bioactivity prior to theadministering, and whereby, as a result of the administering, the GLP-1moiety is released from the conjugate and is effective to result in areduction in blood glucose level in the subject over a period of timeprolonged over that observed for native GLP-1.

In yet another aspect, provided herein is a method of lowering the bloodglucose level in a mammalian subject in need thereof, comprisingadministering to the subject a therapeutically effective amount of aGLP-1 polymer conjugate, to thereby produce lowered blood glucose levelsin the subject over an extended period of at least about 8 hourspost-administering.

In one or more related embodiments, the method comprises administering aGLP-1 polymer conjugate to such a subject, wherein upon administration,the conjugate is hydrolyzed over a period of several days (e.g., 2-7days, 2-6 days, 3-6 days, 3-4 days) to thereby release GLP-1 into thebloodstream. The releasable, e.g., hydrolyzable, conjugates areeffective, in certain embodiments, to lower blood glucose levels over anextended period of greater than about 48 hours (e.g., for about 1-3 daysor so, or for about 1-2.5 days or so).

Also provided herein is the use of a GLP-1 conjugate for the preparationof a medicament to be delivered to the lungs of a mammalian subject,wherein delivery comprises administering the medicament by inhalationfor deposition in and absorption from the lung of the subject, for thetreatment of diabetes.

The present invention further encompasses a method of stimulating theGLP-1 receptor in a mammalian subject. The method comprisesadministering to the subject a therapeutically effective amount of aGLP-1 conjugate. The method may be used to treat subjects having acondition selected from non-insulin dependent diabetes, stress-inducedhyperglycemia, obesity, gastric and/or intestinal motility or emptyingdisorders.

Each of the herein-described features of the invention is meant to applyequally to each and every embodiment as described herein, unlessotherwise indicated.

Additional objects, advantages and novel features of the invention willbe set forth in the description that follows, and in part, will becomeapparent to those skilled in the art upon reading the following, or maybe learned by practice of the invention.

DESCRIPTION OF SEQUENCE LISTING

The following Table describes the amino acid sequences referred to inthe Specification.

Table of Amino Acid Sequences SEQ Name ID NO: Sequence GLP-1 (7-36) 1HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR GLP-1 (7-37) 2HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG Exendin-3 3HSDGTFTSDLSKQMEEEAVRLFIEWLKNGGP SSGAPPPS Exendin-4 4HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGP (C-terminus SSGAPPPS amidated)

BRIEF DESCRIPTION OF THE FIGURES

The present invention is further described in the description ofinvention that follows, in reference to the noted plurality ofnon-limiting drawings, wherein:

FIG. 1 corresponds to an SDS-PAGE analysis of a G2PEG2Fmoc_(20K)-GLP-1reaction mixture as described in Example 3. Lane 1: Invitrogen Mark 12unstained standard. Lane 2: G2PEG2Fmoc_(20K)-N^(ter)-GLP-1 reactionmixture.

FIG. 2 demonstrates the results of purification of monoPEGylatedG2PEG2Fmoc_(20k)-N^(ter)-GLP-1 by cation exchange chromatography asdescribed in Example 3.

FIG. 3 corresponds to an SDS-PAGE analysis of monoPEGylatedG2PEG2Fmoc_(20k)-N^(ter)-GLP-1 before and after the release of GLP-1(Example 3). Lane 1: Invitrogen Mark 12 unstained standard. Lane 2:MonoPEGylated G2PEG2-Fmoc_(20k)-N^(ter)-GLP-1 conjugate followingpurification by ion exchange chromatography. Lane 3: Following completerelease of GLP-1 from the G2PEG2Fmoc_(20k)-N^(rer)-GLP-1 conjugate.

FIGS. 4A, 4B demonstrate a reverse phase HPLC analysis of monoPEGylatedG2PEG2Fmoc_(20k)-N^(ter)-GLP-1 conjugate following purification by ionexchange chromatography (FIG. 4A) and after release of GLP-1 from theG2PEG2Fmoc_(20k)-N^(ter-GLP-)1 conjugate (FIG. 4B), as described inExample 3.

FIG. 5 illustrates the results of purification of monoPEGylatedG2PEG2Fmoc_(40k)-N^(ter)-GLP-1 by cation exchange chromatography asdescribed in Example 4.

FIG. 6 shows the results of an SDS-PAGE analysis of monoPEGylatedG2PEG2Fmoc_(40k)-N^(ter)-GLP-1 before and after release of GLP-1(Example 4). Lane 1: Invitrogen Mark 12 unstained standard. Lane 2:MonoPEGylated G2PEG2Fmoc_(40k)-N^(ter)-GLP-1 conjugate followingpurification by ion exchange chromatography. Lane 3: Following releaseof GLP-1 from the G2PEG2-Fmoc_(40k)-N^(ter)-GLP-1 conjugate.

FIG. 7 demonstrates purification of monoPEGylatedG2PEG2Fmoc_(20k)-Lys-GLP-1 by cation exchange chromatography (Example5).

FIG. 8 corresponds to an SDS-PAGE analysis of monoPEGylatedG2PEG2Fmoc_(20k)-Lys-GLP-1 purified by cation exchange chromatography(Example 5). Lane 1: Invitrogen Mark 12 unstained standard. Lanes 2through 6: Fractions containing monoPEGylated G2PEG2Fmoc_(20k)-Lys-GLP-1conjugate following five individual purifications by ion exchangechromatography.

FIG. 9 illustrates the results of purification of monoPEGylatedG2PEG2Fmoc_(40k)-Lys-GLP-1 by cation exchange chromatography (Example6).

FIG. 10 represents an SDS-PAGE analysis of G2PEG2Fmoc_(40k)-Lys-GLP-1reaction mixture and fractions from one cation exchange chromatographicpurification as described in Example 6. Lane 1: Invitrogen Mark 12unstained standard. Lane 2: Reaction mixture ofG2PEG2Fmoc_(40k)-Lys-GLP-1. Lanes 3-5: Fractions from the peak atretention volume of 9.37 mL. Lanes 6-10: Fractions of monoPEGylatedG2PEG2Fmoc_(40k)-Lys-GLP-1 collected from the peak at retention volumeof 158.3 mL.

FIG. 11 demonstrates the results of purification of monoPEGylatedmPEG_(2k)-Lys-GLP-1 by cation exchange chromatography as described inExample 7.

FIG. 12 corresponds to an SDS-PAGE analysis of purifiedmPEG_(2k)-Lys-GLP-1 (Example 7). Lane 1: MonoPEGylatedmPEG_(2k)-Lys-GLP-1 after chromatographic purification. Lane 2:Invitrogen Mark 12 unstained standard.

FIG. 13 is a plot demonstrating the comparative blood glucose-loweringeffects over time of GLP-1, G2PEG2Fmoc_(20k)-Lys-GLP-1 conjugate andG2PEG2Fmoc_(40k)-Lys-GLP-1 conjugate when subcutaneously administered todb/db mice as described in Example 8.

FIG. 14 is a plot demonstrating the comparative blood glucose-loweringeffects over time of GLP-1, G2PEG2Fmoc_(2k)-N^(ter)-GLP-1 conjugate andG2PEG2Fmoc_(40k)-N^(ter)-GLP-1 conjugate when subcutaneouslyadministered to db/db mice as described in Example 8.

FIG. 15 is a plot showing the in vitro release profile ofG2PEG2Fmoc_(20k)-N^(ter)-GLP-1 as described in Example 21.

FIG. 16 is a plot of group mean blood glucose concentrations in miceadministered exenatide (subcutaneous), PEG2-CAC-Fmoc_(4k)-N^(ter)-GLP-1(intratracheal) or vehicle (intratracheal). Blood glucose concentrationsare expressed as % of value measured at 24 hours pre-dose as describedin Example 25.

FIG. 17 is a plot of group mean blood glucose concentrations in miceadministered exenatide (subcutaneous), mPEGSPC_(5k)-N^(ter)-GLP-1(intratracheal) or vehicle (intratracheal) as described in Example 26.

DESCRIPTION OF THE INVENTION

Before describing one or more embodiments of the present invention indetail, it is to be understood that this invention is not limited to theparticular polymers, synthetic techniques, GLP-1 moieties, and the like,as such may vary.

Unless otherwise stated, a reference to a compound or component includesthe compound or component by itself, as well as in combination withother compounds or components, such as mixtures of compounds.

As used in this specification and the claims, the singular forms “a,”“an,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a polymer” includesa single polymer as well as two or more of the same or differentpolymers, reference to “a pharmaceutically acceptable excipient” refersto a single pharmaceutically acceptable excipient as well as two or moreof the same or different pharmaceutically acceptable excipients, and thelike.

In describing and claiming the present invention(s), the followingterminology will be used in accordance with the definitions providedbelow.

“PEG,” “polyethylene glycol” and “poly(ethylene glycol)” as used herein,are interchangeable. Typically, PEGs for use in accordance with theinvention comprise the following structure: “—(OCH₂CH₂)_(n)—” where (n)is 2 to 4000. As used herein, PEG also includes“—CH₂CH₂—O(CH₂CH₂O)_(n)—CH₂CH₂—” and “—(OCH₂CH₂)_(n)O—,” depending uponwhether or not the terminal oxygens have been displaced. When the PEGfurther comprises a spacer moiety or a linker covalently attachedthereto (to be described in greater detail below), the atoms comprisingthe spacer moiety or linker, when covalently attached to PEG, do notresult in the formation of an oxygen-oxygen bond (i.e., an “—O—O—” orperoxide linkage). Throughout the specification and claims, it should beremembered that the term “PEG” includes structures having variousterminal or “end capping” groups. The term “PEG” also means a polymerthat contains a majority, that is to say, greater than 50%, of —OCH₂CH₂—or —CH₂CH₂O-repeating subunits. With respect to specific forms, the PEGcan take any number of a variety of molecular weights, as well asstructures or geometries such as “branched,” “linear,” “forked,”“multifunctional,” and the like, to be described in greater detailherein.

The terms “end-capped” and “terminally capped” are interchangeably usedherein to refer to a terminal or endpoint of a polymer having anend-capping moiety. Typically, although not necessarily, the end-cappingmoiety comprises a hydroxy or C₁₋₂₀ alkoxy group, such as a C₁₋₁₀ alkoxygroup or a C₁₋₅ alkoxy group. Thus, examples of end-capping moietiesinclude alkoxy (e.g., methoxy, ethoxy), benzyloxy, as well as aryl,heteroaryl, cyclo, heterocyclo, and the like. The end-capping moiety mayinclude one or more atoms of the terminal monomer in the polymer [e.g.,the end-capping moiety “methoxy” in CH₃(OCH₂CH₂)_(n)—]. In addition,saturated, unsaturated, substituted and unsubstituted forms of each ofthe foregoing are envisioned. Moreover, the end-capping group can alsobe a silane. The end-capping group can also advantageously comprise adetectable label. When the polymer has an end-capping group comprising adetectable label, the amount and/or location of the polymer and/or themoiety (e.g., active agent) to which the polymer is coupled can bedetermined by using a suitable detector. Such labels include, withoutlimitation, fluorescers, chemiluminescers, moieties used in enzymelabeling, colorimetric moieties (e.g., dyes), metal ions, radioactivemoieties, and the like. Suitable detectors include photometers, films,spectrometers, and the like.

“Non-naturally occurring” with respect to a polymer as described herein,means a polymer that in its entirety is not found in nature, i.e., issynthetic. A non-naturally occurring polymer may, however, contain oneor more monomers or segments of monomers that are naturally occurring,so long as the overall polymer structure is not found in nature.

The term “water soluble” as in a “water-soluble polymer” is any polymerthat is soluble in water at room temperature. The water-soluble polymeris a polymer having a solubility of 1% (w/v) or more in water at 25° C.Typically, a water-soluble polymer will transmit at least about 75%,such as at least about 95%, of light transmitted by the same solutionafter filtering. On a weight basis, a water-soluble polymer will oftenbe at least about 35% (w/v) soluble in water, such as at least about 50%(w/v) soluble in water, at least about 70% (w/v) soluble in water, or atleast about 85% (w/v) soluble in water, at 25° C. Typically, thewater-soluble polymer is at least about 95% (w/v) soluble in water orcompletely soluble in water.

“Hydrophilic”, e.g, in reference to a “hydrophilic polymer”, refers to apolymer that is characterized by its solubility in and compatabilitywith water. In non-cross linked form, a hydrophilic polymer is able todissolve in, or be dispersed in water. Typically, a hydrophilic polymerpossesses a polymer backbone composed of carbon and hydrogen, andgenerally possesses a high percentage of oxygen in either the mainpolymer backbone or in pendent groups substituted along the polymerbackbone, thereby leading to its “water-loving” nature. Thewater-soluble polymers of the present invention are typicallyhydrophilic, e.g., non-naturally occurring hydrophilic.

Molecular weight in the context of a water-soluble polymer, such as PEG,can be expressed as either a number-average molecular weight or aweight-average molecular weight. Unless otherwise indicated, allreferences to molecular weight herein refer to the weight-averagemolecular weight. Both molecular weight determinations, number-averageand weight-average, can be measured using gel permeation chromatographicor other liquid chromatographic techniques. Other methods for measuringmolecular weight values can also be used, such as the use of end-groupanalysis or the measurement of colligative properties (e.g.,freezing-point depression, boiling-point elevation, or osmotic pressure)to determine number-average molecular weight or the use of lightscattering techniques, ultracentrifugation or viscometry to determineweight-average molecular weight. The polymers of the invention aretypically polydisperse (i.e., number-average molecular weight andweight-average molecular weight of the polymers are not equal),possessing low polydispersity values such as less than about 1.2, lessthan about 1.15, less than about 1.10, less than about 1.05, and lessthan about 1.03. As used herein, references will at times be made to asingle water-soluble polymer having either a weight-average molecularweight or number-average molecular weight; such references will beunderstood to mean that the single water-soluble polymer was obtainedfrom a composition of water-soluble polymers having the stated molecularweight.

The terms “active” or “activated” when used in conjunction with aparticular functional group, refer to a reactive functional group thatreacts readily with an electrophile or a nucleophile on anothermolecule. This is in contrast to those groups that require strongcatalysts or highly impractical reaction conditions in order to react(i.e., a “non-reactive” or “inert” group).

As used herein, the term “functional group” or any synonym thereof ismeant to encompass protected forms thereof as well as unprotected forms.

The terms “spacer moiety,” “linkage,” or “linker” are used herein torefer to an atom or a collection of atoms used to link interconnectingmoieties such as a terminus of a polymer and a GLP-1 moiety or anelectrophile or nucleophile of a GLP-1 moiety. The spacer moiety may behydrolytically stable or may include a physiologically hydrolyzable orenzymatically degradable linkage.

“Alkyl” refers to a hydrocarbon chain, typically ranging from about 1 to15 atoms in length. Such hydrocarbon chains are often not necessarilysaturated and may be branched or straight chain. Straight chain istypical. Exemplary alkyl groups include methyl, ethyl, propyl, butyl,pentyl, 1-methylbutyl, 1-ethylpropyl, 3-methylpentyl, and the like. Asused herein, “alkyl” includes cycloalkyl as well ascycloalkylene-containing alkyl.

“Lower alkyl” refers to an alkyl group containing from 1 to 6 carbonatoms, and may be straight chain or branched. Nonlimiting examples oflower alkyl include methyl, ethyl, n-butyl, i-butyl, and t-butyl.

“Cycloalkyl” refers to a saturated or unsaturated cyclic hydrocarbonchain, including bridged, fused, or spiro cyclic compounds, such asthose made up of 3 to about 12 carbon atoms, or 3 to about 8 carbonatoms. “Cycloalkylene” refers to a cycloalkyl group that is insertedinto an alkyl chain by bonding of the chain at any two carbons in thecyclic ring system.

“Alkoxy” refers to an —O—R group, wherein R is alkyl or substitutedalkyl, such as C₁₋₆ alkyl (e.g., methoxy, ethoxy, propyloxy, and soforth).

The term “substituted” as in, for example, “substituted alkyl,” refersto a moiety (e.g., an alkyl group) substituted with one or morenoninterfering substituents, such as, but not limited to: alkyl, C₃₋₈cycloalkyl, e.g., cyclopropyl, cyclobutyl, and the like; halo, e.g.,fluoro, chloro, bromo, and iodo; cyano; alkoxy; lower phenyl;substituted phenyl; and the like. “Substituted aryl” is aryl having oneor more noninterfering substituents. For substitutions on a phenyl ring,the substituents may be in any orientation (i.e., ortho, meta, or para).

“Noninterfering substituents” are those groups that, when present in amolecule, are typically nonreactive with other functional groupscontained within the molecule.

“Aryl” means one or more aromatic rings, each of 5 or 6 core carbonatoms. Aryl includes multiple aryl rings that may be fused, as innaphthyl or unfused, as in biphenyl. Aryl rings may also be fused orunfused with one or more cyclic hydrocarbon, heteroaryl, or heterocyclicrings. As used herein, “aryl” includes heteroaryl and heterocycle.

“Heteroaryl” is an aryl group containing from one to four heteroatoms,such as sulfur, oxygen, or nitrogen, or a combination thereof.Heteroaryl rings may also be fused with one or more cyclic hydrocarbon,heterocyclic, aryl, or heteroaryl rings. As used herein, “heteroaryl”includes substituted heteroaryl.

“Heterocycle” or “heterocyclic” means one or more rings of 5-12 atoms,such as 5-7 atoms, with or without unsaturation or aromatic characterand having at least one ring atom that is not a carbon. Heteroatomsinclude, but are not limited to, sulfur, oxygen, and nitrogen. As usedherein, “heterocycle” includes substituted heterocycle.

“Substituted heteroaryl” is heteroaryl having one or more noninterferinggroups as substituents.

“Substituted heterocycle” is a heterocycle having one or more sidechains formed from noninterfering substituents.

An “organic radical” as used herein shall include alkyl, substitutedalkyl, aryl, and substituted aryl.

“Electrophile” and “electrophilic group” refer to an ion or atom orcollection of atoms that may be ionic, having an electrophilic center,i.e., a center that is electron seeking, capable of reacting with anucleophile.

“Nucleophile” and “nucleophilic group” refer to an ion or atom orcollection of atoms that may be ionic having a nucleophilic center,i.e., a center that is seeking an electrophilic center or capable ofreacting with an electrophile.

A “hydrolytically degradable” or “hydrolyzable” linkage or bond is abond that reacts with water (i.e., is hydrolyzed) under physiologicalconditions. Examples include bonds that have a hydrolysis half-life atpH 8, 25° C. of less than about 30 minutes. The tendency of a bond tohydrolyze in water will depend not only on the general type of linkageconnecting two given atoms but also on the substituents attached to thetwo given atoms. Hydrolytically unstable or degradable linkages includebut are not limited to carbamate, carboxylate ester (referred to hereinsimply as “ester”), phosphate ester, anhydrides, acetals, ketals,acyloxyalkyl ether, imine, orthoester, peptide and oligonucleotide.Hydrolytically degradable linkages exclude linkages in which cleavage ofa carrier group becomes effective only after unmasking an activatinggroup, such as disclosed in WO 2005/099768, which is incorporated hereinby reference in its entirety. In other words, hydrolytically degradablelinkages exclude linkages based on cascade cleavage mechanisms.

“Releasably attached”, e.g., in reference to a GLP-1 moiety releasablyattached to a water-soluble polymer, refers to a moiety such as a GLP-1moiety that is covalently attached via a linker that includes ahydrolytically degradable linkage as defined above, wherein uponhydrolysis, the GLP-1 moiety is released. The GLP-1 moiety thus releasedwill typically correspond to the unmodified parent or native GLP-1moiety, or may be slightly altered, e.g., to possess a short organic tagof no more than about 8 atoms or so, e.g., typically resulting fromincomplete cleavage of the water-soluble polymer. Preferably, theunmodified parent GLP-1 moiety is released. In instances in which thewater-soluble polymer includes or is covalently attached to the GLP-1moiety via a linker comprising an aryl group, release of the GLP-1moiety occurs via a mechanism which involves neither a 1,4- nor a1,6-elimination step.

An “enzymatically degradable linkage” means a linkage that is subject todegradation by one or more enzymes.

A “hydrolytically stable” linkage or bond refers to a chemical bond,typically a covalent bond, which is substantially stable in water, thatis to say, does not undergo hydrolysis under physiological conditions toany appreciable extent over an extended period of time. Examples ofhydrolytically stable linkages include, but are not limited to, thefollowing: carbon-carbon bonds (e.g., in aliphatic chains), ethers,amides, urethane, and the like. Generally, a hydrolytically stablelinkage is one that exhibits a rate of hydrolysis of less than about1-2% per day under physiological conditions. Hydrolysis rates ofrepresentative chemical bonds can be found in most standard chemistrytextbooks.

“Pharmaceutically acceptable excipient” refers to an excipient that mayoptionally be included in a composition and that causes no significantadverse toxicological effects to a patient upon administration.

A “pharmaceutical composition” is a composition useful forpharmaceutical purposes.

As used herein, “effective amount” refers to an amount covering boththerapeutically effective amounts and prophylactically effectiveamounts.

As used herein, “therapeutically effective amount” refers to an amountthat is effective to achieve the desired therapeutic result. Forexample, an effective amount of a pharmaceutical composition to treat orameliorate diabetes is an amount sufficient to reduce or eliminate thesymptoms of diabetes, for example, an amount that is needed to provide adesired level of insulin in the bloodstream to result in reduced bloodglucose. The pharmacologically effective amount of a givenpharmaceutical composition will vary with factors such as the nature ofthe active component in the composition, the route of administration,the size and species of the animal to receive the composition, and thepurpose of the administration. The suitable amount can be readilydetermined by one skilled in the art based upon available literature andthe information provided herein.

As used herein, “prophylactically effective amount” refers to an amountthat is effective to achieve the desired prophylactic result. Because aprophylactic dose is administered in patients prior to onset of disease,the prophylactically effective amount typically is less than thetherapeutically effective amount.

“Multi-functional” means a polymer having three or more functionalgroups contained therein, where the functional groups may be the same ordifferent. Multi-functional polymeric reagents will typically containfrom about 3-100 functional groups, or from 3-50 functional groups, orfrom 3-25 functional groups, or from 3-15 functional groups, or from 3to 10 functional groups, or will contain 3, 4, 5, 6, 7, 8, 9, or 10functional groups within the polymer backbone.

The term “GLP-1 moiety,” as used herein, refers to a moiety having GLP-1activity. The GLP-1 moiety will also have at least one electrophilicgroup or nucleophilic group suitable for reaction with a water-solublepolymer as provided herein. In addition, the term “GLP-1 moiety”encompasses both the GLP-1 moiety prior to conjugation as well as theGLP-1 moiety residue following conjugation. As will be explained infurther detail below, one of ordinary skill in the art can determinewhether any given moiety has GLP-1 activity. As used herein, the term“GLP-1 moiety” includes peptides modified deliberately, as for example,by site directed mutagenesis or accidentally through mutations. The term“GLP-1 moiety” includes native GLP-1 (GLP-1 (7-37)OH or GLP-1(7-36)NH₂), GLP-1 analogs, GLP-1 derivatives, GLP-1 biologically activefragments, extended GLP-1 (see, for example, International PatentPublication No. WO 03/058203, which is incorporated herein by reference,in particular with respect to the extended glucagon-like peptide-1analogs described therein), and exendin-4 analogs and exendin-4derivatives comprising one or two cysteine residues at particularpositions within GLP-1 as described in WO 2004/093823, which isincorporated herein by reference.

“GLP-1” refers to a compound having GLP-1 activity. As used herein, theterm “GLP-1” includes peptides modified deliberately, as for example, bysite directed mutagenesis or accidentally through mutations. The term“GLP-1” includes native GLP-1 (GLP-1 (7-37)OH or GLP-1 (7-36)NH₂), GLP-1analogs, GLP-1 derivatives, GLP-1 biologically active fragments,extended GLP-1 (see, for example, International Patent Publication No.WO 03/058203, which is incorporated herein by reference, in particularwith respect to the extended glucagon-like peptide-1 analogs describedtherein), and exendin-4 analogs and exendin-4 derivatives comprising oneor two cysteine residues at particular positions within GLP-1 asdescribed in WO 2004/093823, which is incorporated herein by reference.

The term “fragment” means any peptide having the amino acid sequence ofa portion of a GLP-1 moiety that retains some degree of GLP-1 activity.Fragments include peptides produced by proteolytic degradation of theGLP-1 or produced by chemical synthesis by methods routine in the art.Often, GLP-1 fragments are obtained after truncation of one or moreamino acids from the N-terminus and/or C-terminus of a GLP-1 moiety, andpossess a degree of GLP-1 activity.

The term “substantially homologous” means that a particular subjectsequence, for example, a mutant sequence, varies from a referencesequence by one or more substitutions, deletions, or additions, the neteffect of which does not result in an adverse functional dissimilaritybetween the reference and subject sequences. For purposes of the presentinvention, sequences having greater than 95 percent homology, equivalentbiological properties (although potentiality different degrees ofactivity), and equivalent expression characteristics are consideredsubstantially homologous. For purposes of determining homology,truncation of the mature sequence should be disregarded. Sequenceshaving lesser degrees of homology, comparable bioactivity, andequivalent expression characteristics are considered substantialequivalents. Exemplary GLP-1 moieties for use herein include thosepeptides having a sequence that is substantially homologous to, e.g.,native GLP-1.

A “deletion variant” of a GLP-1 moiety is a peptide in which one or moreamino acid residues of the GLP-1 moiety have been deleted and the aminoacid residues preceding and following the deleted amino acid residue areconnected via an amide bond (except in instances where the deleted aminoacid residue was located on a terminus of the peptide or protein).Deletion variants include instances where only a single amino acidresidue has been deleted, as well as instances where two amino acids aredeleted, three amino acids are deleted, four amino acids are deleted,and so forth. Each deletion retains some degree of GLP-1 activity.

A “substitution variant” of a GLP-1 moiety is peptide or protein inwhich one or more amino acid residues of the GLP-1 moiety have beendeleted and a different amino acid residue has taken its place.Substitution variants include instances where only a single amino acidresidue has been substituted, as well as instances where two amino acidsare substituted, three amino acids are substituted, four amino acids aresubstituted, and so forth. Each substitution variant has some degree ofGLP-1 activity.

An “addition variant” of a GLP-1 moiety is a peptide in which one ormore amino acid residues of the GLP-1 have been added into an amino acidsequence and adjacent amino acid residues are attached to the addedamino acid residue by way of amide bonds (except in instances where theadded amino acid residue is located on a terminus of the peptide,wherein only a single amide bond attaches the added amino acid residue).Addition variants include instances where only a single amino acidresidue has been added, as well as instances where two amino acids areadded, three amino acids are added, four amino acids are added, and soforth. Each addition variant has some degree of GLP-1 activity.

“Insulinotropic activity” refers to the ability to stimulate insulinsecretion in response to elevated glucose levels, thereby causingglucose intake by cells and decreased plasma glucose levels.Insulinotropic activity can be measured by methods known in the art,such as by measuring GLP-1 receptor binding activity or activation,e.g., assays employing pancreatic islet cells or insulinoma cells, asdescribed in EP 619 322 and U.S. Pat. No. 5,120,712, respectively, whichare incorporated herein by reference. Insulinotropic activity istypically measured in humans by measuring insulin or C-peptide levels.

The terms “subject”, “individual,” or “patient” are used interchangeablyherein and refer to a vertebrate, preferably a mammal. Mammals include,but are not limited to, murines, rodents, simians, humans, farm animals,sport animals, and pets.

“Optional” or “optionally” means that the subsequently describedcircumstance may or may not occur, so that the description includesinstances where the circumstance occurs and instances where it does not.

“Substantially” (unless specifically defined for a particular contextelsewhere or the context clearly dictates otherwise) means nearlytotally or completely, for instance, satisfying one or more of thefollowing: greater than 50%, 51% or greater, 75% or greater, 80% orgreater, 90% or greater, and 95% or greater of the condition.

Unless the context clearly dictates otherwise, when the term “about”precedes a numerical value, the numerical value is understood to mean±10% of the stated numerical value.

“Amino acid” refers to any compound containing both an amino group and acarboxylic acid group. Although the amino group most commonly occurs atthe position adjacent to the carboxy function, the amino group may bepositioned at any location within the molecule. The amino acid may alsocontain additional functional groups, such as amino, thio, carboxyl,carboxamide, imidazole, etc. An amino acid may be synthetic or naturallyoccurring, and may be used in either its racemic or optically active(D-, or L-) form.

Amino acid residues in peptides are abbreviated as follows:Phenylalanine is Phe or F; Leucine is Leu or L; Isoleucine is Ile or I;Methionine is Met or M; Valine is Val or V; Serine is Ser or S; Prolineis Pro or P; Threonine is Thr or T; Alanine is Ala or A; Tyrosine is Tyror Y; Histidine is His or H; Glutamine is Gln or Q; Asparagine is Asn orN; Lysine is Lys or K; Aspartic Acid is Asp or D; Glutamic Acid is Gluor E; Cysteine is Cys or C; Tryptophan is Trp or W; Arginine is Arg orR; and Glycine is Gly or G.

A “sustained release composition” or an “extended release composition”is a composition that releases the active component slowly over arelatively longer period of time than an “immediate release”composition. In general, the active component, e.g., GLP-1 polymerconjugate, is released over at least about 3 hours, or at least about 4hours, or at least about 5 hours, or at least about 6 hours, or at leastabout 8 hours.

A “sustained plasma level” of a protein for a specified period of timemeans that the protein can be detected in the plasma for a durationspecified. A protein can be detected by any methods for detecting suchprotein, e.g., immunological, biochemical, or functional methods. Forexample, insulin can be detected by enzyme-linked immunosorbent assay(ELISA), mass spectrometry, or determination of blood glucose levels.

A composition that is “suitable for pulmonary delivery” refers to acomposition that is capable of being aerosolized and inhaled by asubject so that a portion of the aerosolized particles reaches the lungsto permit penetration into the alveoli. Such a composition may beconsidered “respirable” or “inhalable.”

An “aerosolized” composition contains liquid or solid particles that aresuspended in a gas (typically air), typically as a result of actuation(or firing) of an inhalation device such as a dry powder inhaler, anatomizer, a metered dose inhaler, or a nebulizer.

A “jet nebulizer” is a system, such as a device, that forces compressedair through a solution of a drug so that a fine spray can be deliveredto a facemask and inhaled. Nebulizers often are used to administer drugsto those who lack the ability to use a metered-dose or breath-activatedinhaler.

A “dry powder inhaler” is a device that is loaded with a unit dosage ofthe drug in powder form. Generally, the inhaler is activated by taking abreath. For example, a capsule or blister is punctured and the powder isdispersed so that it can be inhaled in, e.g., a “Spinhaler” or“Rotahaler.” “Turbohalers” are fitted with canisters that delivermeasured doses of the drug in powder form.

A “metered dose inhaler” or “MDI” is a device that delivers a measureddose of a drug in the form of a suspension of extremely small liquid orsolid particles, which is dispensed from the inhaler by a propellantunder pressure. Such inhalers are placed into the mouth and depressed(activated) to release the drug as the individual takes a breath.

As used herein, the term “emitted dose” or “ED” refers to an indicationof the delivery of dry powder from an inhaler device after an actuationor dispersion event from a powder unit or reservoir. ED is defined asthe ratio of the dose delivered by an inhaler device to the nominal dose(i.e., the mass of powder per unit dose placed into a suitable inhalerdevice prior to firing). The ED is an experimentally determined amount,and may be determined using an in vitro device set-up which mimicspatient dosing. To determine an ED value, as used herein, dry powder isplaced into a Pulmonary Delivery System (PDS) device (NektarTherapeutics), described in U.S. Pat. No. 6,257,233, which isincorporated herein by reference in its entirety. The PDS device isactuated, dispersing the powder. The resulting aerosol cloud is thendrawn from the device by vacuum (30 L/min) for 2.5 seconds afteractuation, where it is captured on a tared glass fiber filter (Gelman,47 mm diameter) attached to the device mouthpiece. The amount of powderthat reaches the filter constitutes the delivered dose. For example, fora blister containing 5 mg of dry powder that is placed into aninhalation device, if dispersion of the powder results in the recoveryof 4 mg of powder on a tared filter as described above, then the ED forthe dry powder composition is 80% (=4 mg (delivered dose)/5 mg (nominaldose)).

A composition in “dry powder form” is a powder composition thattypically contains less than about 20 wt %, less than about 10 wt %, orless than about 5 wt %, of water.

As used herein, “mass median diameter” or “MMD” refers to the mediandiameter of a plurality of particles, typically in a polydisperseparticle population, i.e., consisting of a range of particle sizes. MMDvalues as reported herein are determined by laser diffraction (SympatecHelos, Clausthal-Zellerfeld, Germany), unless the context indicatesotherwise. Typically, powder samples are added directly to the feederfunnel of the Sympatec RODOS dry powder dispersion unit. This can beachieved manually or by agitating mechanically from the end of a VIBRIvibratory feeder element. Samples are dispersed to primary particles viaapplication of pressurized air (2 to 3 bar), with vacuum depression(suction) maximized for a given dispersion pressure. Dispersed particlesare probed with a 632.8 nm laser beam that intersects the dispersedparticles' trajectory at right angles. Laser light scattered from theensemble of particles is imaged onto a concentric array ofphotomultiplier detector elements using a reverse-Fourier lens assembly.Scattered light is acquired in time-slices of 5 ms. Particle sizedistributions are back-calculated from the scattered lightspatial/intensity distribution using an algorithm.

“Mass median aerodynamic diameter,” or “MMAD,” is a measure of theaerodynamic size of a dispersed particle. The aerodynamic diameter isused to describe an aerosolized powder in terms of its settlingbehavior, and is the diameter of a unit density sphere having the samesettling velocity, in air, as the particle. The aerodynamic diameterencompasses particle shape, density, and physical size of a particle. Asused herein, MMAD refers to the midpoint or median of the aerodynamicparticle size distribution of an aerosolized powder determined bycascade impaction at standard conditions using a Pulmonary DeliverySystem (PDS) device (Nektar Therapeutics), described in U.S. Pat. No.6,257,233, which is incorporated herein by reference in its entirety,unless otherwise indicated.

“Fine particle fraction” is the fraction of particles with anaerodynamic diameter that is less than 5 microns. Where specified, thefine particle fraction may also refer to the fraction of particles withan aerodynamic diameter that is less than 3.3 microns.

“Treating or ameliorating” a disease or medical condition means reducingor eliminating the symptoms of the disease or medical condition. In someembodiments, “treating or ameliorating” a disease or medical conditionwill be directed at addressing the cause of the disease or medicalcondition. Treating a disease may result in cure of the disease.

“Basal insulin” refers to a long-acting insulin that is sufficient tosustain steady low levels of insulin effective to satisfy basalrequirements. Generally, a basal insulin is one exhibiting a prolongedtime of action of greater than about 8 hours in a standard model ofdiabetes. Commercially available basal insulins include neutralprotamine Hagedorn (NPH), Lente, and Ultralente'—and analogs such asinsulin glargine.

A GLP-1 polymer conjugate which lacks bioactivity is one which, whenevaluated in an assay suitable for assessing GLP-1 bioactivity,possesses a bioactivity of less than about 2% when compared to nativeGLP-1. Various assays may be used to assess bioactivity, includingin-vitro and in-vivo assays that measure GLP-1 receptor binding activityor receptor activation, as described in greater detail herein. Areceptor-signaling assay may also be used to assess GLP-1 activity (see,e.g., Zlokarnik et al, Science, 1998, 279:84-88).

A “monomer” or “mono-conjugate”, in reference to a polymer conjugate ofGLP-1, refers to a GLP-1 moiety having only one water-soluble polymermolecule covalently attached thereto, whereas a GLP-1 “dimer” or“di-conjugate” is a polymer conjugate of GLP-1 having two water-solublepolymer molecules covalently attached thereto, and so forth.

Turning now to one or more aspects of the present invention, conjugatesare provided, the conjugates typically comprising a GLP-1 moietyreleasably attached, either directly via a hydrolyzable linkage or via alinker comprised of a hydrolyzable linkage, to a water-soluble polymer.The conjugates of the invention, when administered in vivo, are usuallyeffective not only in providing a significant reduction in blood glucoselevels, but have been shown to possess extended release properties. Thatis to say, the blood glucose lowering ability of the present conjugatesis typically effective over a duration of hours, and in some instances,days, in contrast to the rapid clearance and short activity of nativeGLP-1. Thus, the conjugates of the invention typically provide severalnotable advantages over native GLP-1. Finally, although it has beensuggested that N-terminal modification of GLP-1 is ineffective toprovide a GLP-1 derivative suitable for pharmacological use, see forexample, International Patent Publication No. WO 04/078777, the presentinvention provides, in one aspect, N-terminally modified water-solublepolymer conjugates that are not only effective, but indeed possessseveral advantages over native GLP-1, to be described in greater detailbelow.

The GLP-1 Moiety

The conjugates of the invention comprise a GLP-1 moiety. As previouslystated, the term “GLP-1 moiety” is meant to encompass the GLP-1 moietyprior to conjugation as well as following attachment to one or morewater-soluble polymers. It will be understood, however, that when theGLP-1 moiety is covalently attached to a water-soluble polymer, theGLP-1 moiety is slightly altered due to the presence of one or morecovalent bonds associated with linkage to the polymer (or linker that isattached to the polymer), due to reaction of one of more reactive groupsof the GLP-1 moiety (e.g., an amino, carboxyl, etc.), with thewater-soluble polymer. Often, this slightly altered form of the GLP-1moiety attached to another molecule, such as a water-soluble polymer, isreferred to as a “residue” of the GLP-1 moiety.

A GLP-1 moiety for use in the invention is any GLP-1 moiety having GLP-1activity. Numerous GLP-1 analogs, derivatives, and variants have beenpreviously described, and are encompassed by the term, “GLP-1 moiety”.One preferred GLP-1 moiety for use in the invention is human GLP-1, a 37amino acid peptide. Its naturally-occurring forms includeGLP-1(7-36)NH₂, GLP-1 (7-37)OH and GLP-1 (7-37)NH₂, where, according toconventional numbering, the N-terminal histidine is assigned as residue7. Commercially-available forms also include GLP-1 (1-36).

The amino acid sequence of GLP-1(7-36) corresponds to: (SEQ ID NO:1).His7-Ala8-Glu9-Gly10-Thr11-Phe12-Thr13-Ser14-Asp15-Val16-Ser17-Ser18-Tyr19-Leu20-Glu21-Gly22-Gln23-Ala24-Ala25-Lys26-Glu27-Phe28-Ile29-Ala30-Trp31-Leu32-Val33-Lys34-Gly35-Arg36

The amino acid sequence of GLP-1 (7-37)OH corresponds to: (SEQ ID NO:2).His7-Ala8-Glu9-Gly10-Thr11-Phe12-Thr13-Ser14-Asp15-Val16-Ser17-Ser18-Tyr19-Leu20-Glu21-Gly22-Gln23-Ala24-Ala25-Lys26-Glu27-Phe28-Ile29-Ala30-Trp31-Leu32-Val33-Lys34-Gly35-Arg36-Gly37

Additional GLP-1 moieties for use in the invention include GLP-1 peptideanalogs such as those described in WO 91/11457, N-terminal truncatedfragments of GLP-1 as described in EP 0 699 686, and cysteine-insertedGLP-1 sequences as described in WO 2004/093823, among others.Cysteine-inserted GLP-1 variants for use in the invention include thosehaving a cysteine inserted at positions selected from amino acid 11, 12,16, 22, 23, 24, 25, 26, 27, 30, 34, 35, 36, and 37 of GLP-1. Suchvariants are typically designated as follows. For instance, a GLP-1variant having a cysteine inserted at positions 22 and 35 is typicallydescribed as [Cys²²Cys³⁵]GLP-1(7-37). Variants for use in preparing aconjugate of the invention often have no more than 1 or 2 cysteine aminoacids per GLP-1 moiety. Exemplary locations for insertion of a cysteineinclude, but are not limited to, amino acids 22, 26, 34, 35, 36, and 37of GLP-1. Such GLP-1 moieties are particularly useful for conjugation toa thiol-selective polymer reagent, such as a water-soluble polymerhaving one or more reactive maleimide functional groups.

Other GLP-1 moieties suitable for use in the invention include GLP-1moieties as described in U.S. Published Application No. 2004/0235710,corresponding to Ser. No. 10/486,333.

Also for use in the present invention are GLP-1 analogs referred to asexendins. Exendins are peptides that were first isolated from thesalivary secretions of the Gila-monster and the Mexican Beaded Lizard.The exendins have a degree of similarity to several members of the GLPfamily, with the highest homology, 53%, to GLP-1(7-36)NH₂ (Goke, et al.,J. Biol. Chem., 268:19650-55, 1993).

Particular exendins for use in the present invention include exendin-3and exendin-4 (synthetic extendin-4 is also known as Exenatide).

Exendin-3 (1-39) has the following amino acid sequence: (SEQ ID NO:3).

His Ser Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu Glu AlaVal Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser Ser Gly Ala ProPro Pro Ser.

The amino acid sequence of exendin-4 (1-39) corresponds to: (SEQ IDNO:4).

His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu Glu AlaVal Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser Ser Gly Ala ProPro Pro Ser

wherein the C-terminus serine is amidated.

The GLP-1 moiety may be obtained from either non-recombinant methods orfrom recombinant methods, and the invention is not limited in thisregard. Moreover, several GLP-1 moieties are commercially available,e.g., hGLP-1, rExendin-4, and rHuGLP-1 are available from ProSpecTanyTechno Gene LTD (Rehovot, Israel); and (Ser8)GLP-1(7-36)amide,hGLP-1amide, hGLP-1(7-36)Lys(biotin)amide, (American Peptide Co.,Sunnyvale, Calif.), among others. Methods for preparing GLP-1 moietiesare well-known, and are described, e.g., in U.S. Pat. Nos. 5,118,666;5,120,712; and 5,523,549.

GLP-1 moieties can be prepared using standard methods of solution orsolid phase peptide synthesis such as those described in Dugas H.,Penny, C., Bioorganic Chemistry, Springer Verlag, New York, p. 54-92(1981); Merrifield, J. M., Chem Soc., 85, 2149 (1962), and Stewart andYoung, Solid Phase Peptide Synthesis, Freeman, San Francisco, p. 24-66(1969). Peptide synthesizers are available from, e.g., AppliedBiosystems, Foster City, Calif. Solid phase synthesizers are typicallyused according to manufacturers' instructions for blocking interferinggroups, protecting certain amino acids, coupling, decoupling, andcapping unreacted amino acids. BOC-amino acids and other reagents arecommercially available from Applied Biosystems. As a non-limitingexample, sequential BOC chemistry using double coupling protocols areapplied to the starting p-methyl benzhydryl amine resins for theproduction of C-terminal carboxamides. For the production of C-terminalacids, the corresponding PAM resin is used. Asn, Gln, and Arg arecoupled using preformed hydroxybenzotriazole esters. Suitable side chainprotecting groups include: Arg (tosyl), Asp (cyclohexyl), Glu(cyclohexyl), Ser (benzyl), Thr (benzyl), and Tyr (4-bromocarbobenzoxy).BOC deprotection may be carried out with trifluoroacetic acid inmethylene chloride. Following synthesis, the resulting peptide may bedeprotected and cleaved from the resin using, e.g., anhydrous HFcontaining 10% meta-cresol.

Exemplary recombinant methods used to prepare a GLP-1 moiety (whether ahuman GLP-1 or a different protein having GLP-1 activity) include thefollowing, among others as will be apparent to one skilled in the art.Typically, a GLP-1 moiety as described herein is prepared byconstructing the nucleic acid encoding the desired polypeptide orfragment, cloning the nucleic acid into an expression vector,transforming a host cell (e.g., plant, bacteria such as Escherichiacoli, yeast such as Saccharomyces cerevisiae, or mammalian cell such asChinese hamster ovary cell or baby hamster kidney cell), and expressingthe nucleic acid to produce the desired polypeptide or fragment. Theexpression can occur via exogenous expression (when the host cellnaturally contains the desired genetic coding) or via endogenousexpression. Methods for producing and expressing recombinantpolypeptides in vitro and in prokaryotic and eukaryotic host cells areknown to those of ordinary skill in the art. See, for example, U.S. Pat.No. 4,868,122.

To facilitate identification and purification of the recombinantpolypeptide, nucleic acid sequences that encode an epitope tag or otheraffinity binding sequence can be inserted or added in-frame with thecoding sequence, thereby producing a fusion protein comprised of thedesired polypeptide and a polypeptide suited for binding. Fusionproteins can be identified and purified by first running a mixturecontaining the fusion protein through an affinity column bearing bindingmoieties (e.g., antibodies) directed against the epitope tag or otherbinding sequence in the fusion proteins, thereby binding the fusionprotein within the column. Thereafter, the fusion protein can berecovered by washing the column with the appropriate solution (e.g.,acid) to release the bound fusion protein. The recombinant polypeptidecan also be identified and purified by lysing the host cells, separatingthe polypeptide, e.g., by size exclusion chromatography, and collectingthe polypeptide. These and other methods for identifying and purifyingrecombinant polypeptides are known to those of ordinary skill in theart. In one or more embodiments of the present invention, the GLP-1moiety is not in the form of a fusion protein. See, for example, Dillonet al., Cloning and Functional Expression of the Human Glucagon-LikePeptide-1 Receptor, Endocrinology, 133:1907-1910 (1993).

For any given moiety, it is possible to determine whether that moietypossesses GLP-1 activity. Various assays may be used to assessbioactivity, including in-vitro and in-vivo assays that measure GLP-1receptor binding activity or receptor activation. See, for example, EP619 322 and U.S. Pat. No. 5,120,712 for descriptions of assessing GLP-1activity. A receptor-signaling assay may also be used to assess GLP-1activity, such as described in Zlokarnik et al, Science, 279, 84-88,1998.

Other methods known to those of ordinary skill in the art can also beused to determine whether a given moiety has GLP-1 activity. Suchmethods are useful for determining the GLP-1 activity of both the moietyitself (and therefore can be used as a “GLP-1 moiety”), as well as thatof the corresponding polymer-moiety conjugate. For example, one candetermine whether a given moiety is an agonist of the human GLP-1receptor by assessing whether that moiety stimulates the formation ofcAMP in a suitable medium containing the human GLP-1 receptor. Thepotency of such moiety is determined by calculating the EC50 value froma dose response curve. As an example, BHK cells (baby hamster kidneycells) expressing the cloned human GLP-1 receptor can be grown in DMEMmedia containing penicillin, streptomycin, calf serum, and Geneticin.The cells are then washed in phosphate buffered saline and harvested.Plasma membranes are then prepared from the cells by homogenization, andthe homogenate is then centrifuged to produce a pellet. The resultingpellet is suspended by homogenization in a suitable buffer, centrifuged,and then washed. The cAMP receptor assay is then carried out bymeasuring cyclic AMP (cAMP) in response to the test insulinotropicmoiety. cAMP can be quantified using the AlphaScreen™ cAMP Kit (PerkinElmer). Incubations are typically carried out in microtiter plates inbuffer, with addition of, e.g., ATP, GTP, IBMX(3-isobutyl-1-methylxanthine, Tween-20, BSA, acceptor beads, and donorbeads incubated with biotinylated-cAMP. Counting may be carried out,e.g., using the Fusion™ instrument (Perkin Elmer).Concentration-response curves are then plotted for the individualinsulinotropic moieties under evaluation, and their EC₅₀ valuesdetermined.

Nonlimiting examples of GLP-1 moieties include any of SEQ ID NO: 1, SEQID NO: 2 and SEQ ID NO: 3; and SEQ ID NO:4; truncated versions thereof;hybrid variants, and peptide mimetics having GLP-1 activity.Biologically active fragments, deletion variants, substitution variantsor addition variants of any of the foregoing that maintain at least somedegree of GLP-1 activity can also serve as a GLP-1 moiety in theconjugates of the invention.

GLP-1 Modifications

A GLP-1 moiety as described herein may also contain one or moreadditional modifications, e.g., the introduction of one or moreglycosides, or methyl groups.

Methylation

A GLP-1 moiety may also possess one or more methyl or other lower alkylgroups at one or more positions of the GLP-1 sequence. Examples of suchgroups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl,pentyl, etc. Sites of modification include residues 7, 8, 9, and/or 10,with the 7 and/or 9 positions being preferred. Introduction of one ormore methyl groups is expected to modify the dipeptidyl peptidase IV(DPP IV) recognition site, such that the modified GLP-1 is protectedagainst degradation by DPP IV. While not wishing to be bound by theory,it is believed that N-methylation at positions other than 7, 8, 9,and/or 10 may disrupt the helicity of GLP-1 and thereby reduce itsactivity.

Introduction of N-terminal modifications to GLP-1 may be carried out asdescribed by Gallwitz, B., et al., Regulatory Peptides, 86 (1-3),103-111 (2000) or WO 2004/007427, which are incorporated herein byreference in their entireties. Illustrative GLP-1 analogues withalternations at the N-terminus include N-methylated GLP-1 (N-me-GLP-1),alpha-methylated GLP-1 (alpha-me-GLP-1), desamidated GLP-1(desamino-GLP-1), and imidazole-lactic acid substituted GLP-1(imi-GLP-1), among others, and are suitable for use in the presentinvention.

Methods for synthesizing various other analogues of GLP-1, includingmethyl-derivatives as described above, as well examples of suchadditional GLP-1 analogues are described in International PatentPublications WO 00/34332, WO 2004/074315, and WO 2005/058955.

Glycosylation

The GLP-1 moieties described herein may also contain one or moreglycosides. Although any glycoside can be used, the GLP-1 moiety ispreferably modified by introduction of either a monosaccharide, adisaccharide, or a trisaccharide. Although any site on the GLP-1 moietymay be modified by introduction of a saccharide, preferably, thesaccharide is introduced at any one or more of positions 7, 8, or 9 toprotect the peptide against DPP IV proteolysis. Further, additionalglycosides may be introduced, e.g., at any one or more of positions 11,13, 14, 17, 18, 21, 22, 23, 24, 26, and 34 to increase the helicitythrough the central portion of the peptide, as well as provideadditional resistance to proteolysis. Glycosylation may occur on anaturally occurring amino acid. Alternatively, a saccharide may becovalently attached to one or more sites of the GLP-1 moiety, whereinthe one or more sites each comprise one of Asp, Asn, Ser, and Thr thatis substituted at one or more sites selected from the N-terminus (His7),Ala8, Glu9, Thr11, Thr13, Ser14, Ser17, Ser18, Glu21, Gly22, Gln23,Lys26, and Lys34.

Glycosylated GLP-1 moieties may be prepared using conventional Fmocchemistry and solid phase peptide synthesis techniques, e.g., on resin,where the desired protected glycoamino acids are prepared prior topeptide synthesis and then introduced into the peptide chain at thedesired position during peptide synthesis. Thus, the GLP-1 polymerconjugates may be conjugated in vitro. The glycosylation may occurbefore deprotection. Preparation of aminoacid glycosides is described inU.S. Pat. No. 5,767,254, WO 2005/097158, and Doores, K., et al., Chem.Commun., 1401-1403, 2006, which are incorporated herein by reference intheir entireties. For example, alpha and beta selective glycosylationsof serine and threonine residues are carried out using the Koenigs-Knorrreaction and Lemieux's in situ anomerization methodology with Schiffbase intermediates. Deprotection of the Schiff base glycoside is thencarried out using mildly acidic conditions or hydrogenolysis. Acomposition, comprising a glycosylated and pegylated GLP-1 conjugatemade by stepwise solid phase peptide synthesis involving contacting agrowing peptide chain with protected amino acids in a stepwise manner,wherein at least one of the protected amino acids is glycosylated,followed by pegylation, may have a purity of at least about 95%, such asat least about 97%, or at least about 98%, of a single species of theglycosylated and pegylated GLP-1 conjugate.

Monosaccharides that may by used for introduction at one or more aminoacid residues of GLP-1 include glucose (dextrose), fructose, galactose,and ribose. Additional monosaccharides suitable for use includeglyceraldehydes, dihydroxyacetone, erythrose, threose, erythrulose,arabinose, lyxose, xylose, ribulose, xylulose, allose, altrose, mannose,as well as others. Glycosides, such as mono-, di-, and trisaccharidesfor use in modifying a GLP-1 moiety, may be naturally occurring or maybe synthetic.

Disaccharides that may by used for introduction at one or more aminoacid residues of GLP-1 include sucrose, lactose, maltose, trehalose,melibiose, and cellobiose, among others. Trisaccharides includeacarbose, raffinose, and melezitose.

The Water-Soluble Polymer

A conjugate of the invention comprises a GLP-1 moiety attached,preferably but not necessarily releasably, to a water-soluble polymer.The water-soluble polymer is typically hydrophilic, nonpeptidic,nontoxic, non-naturally occurring and biocompatible. A substance isconsidered biocompatible if the beneficial effects associated with useof the substance alone or with another substance (e.g., an active agentsuch a GLP-1 moiety) in connection with living tissues (e.g.,administration to a patient) outweighs any deleterious effects asevaluated by a clinician, e.g., a physician. A substance is considerednonimmunogenic if the intended use of the substance in vivo does notproduce an undesired immune response (e.g., the formation of antibodies)or, if an immune response is produced, that such a response is notdeemed clinically significant or important as evaluated by a clinician.Typically, the water-soluble polymer is hydrophilic, biocompatible andnonimmunogenic.

Further the water-soluble polymer is typically characterized as havingfrom 2 to about 300 termini, preferably from 2 to 100 termini, and morepreferably from about 2 to 50 termini. Examples of such polymersinclude, but are not limited to, poly(alkylene glycols) such aspolyethylene glycol (PEG), poly(propylene glycol) (“PPG”), copolymers ofethylene glycol and propylene glycol and the like, poly(oxyethylatedpolyol), poly(olefinic alcohol), poly(vinylpyrrolidone),poly(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate),poly(saccharides), poly(α-hydroxy acid), poly(vinyl alcohol),polyphosphazene, polyoxazoline, poly(N-acryloylmorpholine), andcombinations of any of the foregoing, including copolymers andterpolymers thereof.

The water-soluble polymer is not limited to a particular structure andmay possess a linear architecture (e.g., alkoxy PEG or bifunctionalPEG), or a non-linear architecture, such as branched, forked,multi-armed (e.g., PEGs attached to a polyol core), or dendritic.Moreover, the the polymer subunits can be organized in any number ofdifferent patterns and can be selected, e.g., from homopolymer,alternating copolymer, random copolymer, block copolymer, alternatingtripolymer, random tripolymer, and block tripolymer.

One particularly preferred type of water-soluble polymer for use in theinvention is a polyalkylene oxide, and in particular, polyethyleneglycol (or PEGs). Generally, a PEG used to prepare a GLP-1 polymerconjugate of the invention is “activated” or reactive. That is to say,the activated PEG (and other activated water-soluble polymerscollectively referred to herein as “polymeric reagents”) used to form aGLP-1 conjugate comprises an activated functional group suitable forcoupling to a desired site or sites on the GLP-1 moiety. Thus, apolymeric reagent for use in preparing a GLP-1 conjugate includes afunctional group for reaction with the GLP-moiety.

Representative polymeric reagents and methods for conjugating suchpolymers to an active moiety are known in the art, and are, e.g.,described in Harris, J. M. and Zalipsky, S., eds, Poly(ethylene glycol),Chemistry and Biological Applications, ACS, Washington, 1997; Veronese,F., and J. M Harris, eds., Peptide and Protein PEGylation, Advanced DrugDelivery Reviews, 54(4); 453-609 (2002); Zalipsky, S., et al., “Use ofFunctionalized Poly(Ethylene Glycols) for Modification of Polypeptides”in Polyethylene Glycol Chemistry: Biotechnical and BiomedicalApplications, J. M. Harris, ed., Plenus Press, New York (1992); Zalipsky(1995) Advanced Drug Reviews 16:157-182, and in Roberts, et al., Adv.Drug Delivery Reviews, 54, 459-476 (2002).

Additional PEG reagents suitable for use in forming a conjugate of theinvention, and methods of conjugation are described in the NektarAdvanced PEGylation Catalogs, 2005-2006; 2004; 2003; and in ShearwaterCorporation, Catalog 2001; Shearwater Polymers, Inc., Catalogs, 2000 and1997-1998, and in Pasut. G., et al., Expert Opin. Ther. Patents (2004),14(5). PEG reagents suitable for use in the present invention alsoinclude those available from NOF Corporation, as described generally onthe NOF website (2006) under Products, High Purity PEGs and ActivatedPEGs. Products listed therein and their chemical structures areexpressly incorporated herein by reference. Additional PEGs for use informing a GLP-1 conjugate of the invention include those available fromPolypure (Norway) and from QuantaBioDesign LTD (Ohio), where thecontents of their online catalogs (2006) with respect to available PEGreagents are expressly incorporated herein by reference.

Typically, the weight-average molecular weight of the water-solublepolymer in the conjugate is from about 100 Daltons to about 150,000Daltons. Exemplary ranges, however, include weight-average molecularweights in the range of from about 250 Daltons to about 80,000 Daltons,from 500 Daltons to about 80,000 Daltons, from about 500 Daltons toabout 65,000 Daltons, from about 500 Daltons to about 40,000 Daltons,from about 750 Daltons to about 40,000 Daltons, from about 1000 Daltonsto about 30,000 Daltons.

For any given water-soluble polymer, a molecular weight in one or moreof these ranges is typical. Generally, a GLP-1 conjugate in accordancewith the invention, when intended for subcutaneous or intravenousadministration, will comprise a PEG or other suitable water-solublepolymer having a weight average molecular weight of about 20,000 Daltonsor greater, while a GLP-1 conjugate intended for pulmonaryadministration will generally, although not necessarily, comprise a PEGpolymer having a weight average molecular weight of about 20,000 Daltonsor less. For example, Examples 25 and 26 provide intratrachealinstillation data in mice for an illustrative branched PEG having anoverall molecular weight of about 8 kD.

Exemplary weight-average molecular weights for the water-soluble polymerinclude about 100 Daltons, about 200 Daltons, about 300 Daltons, about400 Daltons, about 500 Daltons, about 600 Daltons, about 700 Daltons,about 750 Daltons, about 800 Daltons, about 900 Daltons, about 1,000Daltons, about 1,500 Daltons, about 2,000 Daltons, about 2,200 Daltons,about 2,500 Daltons, about 3,000 Daltons, about 4,000 Daltons, about4,400 Daltons, about 4,500 Daltons, about 5,000 Daltons, about 5,500Daltons, about 6,000 Daltons, about 7,000 Daltons, about 7,500 Daltons,about 8,000 Daltons, about 9,000 Daltons, about 10,000 Daltons, about11,000 Daltons, about 12,000 Daltons, about 13,000 Daltons, about 14,000Daltons, about 15,000 Daltons, about 20,000 Daltons, about 22,500Daltons, about 25,000 Daltons, about 30,000 Daltons, about 35,000Daltons, about 40,000 Daltons, about 45,000 Daltons, about 50,000Daltons, about 55,000 Daltons, about 60,000 Daltons, about 65,000Daltons, about 70,000 Daltons, and about 75,000 Daltons.

Branched versions of the water-soluble polymer (e.g., a branched 40,000Dalton water-soluble polymer comprised of two 20,000 Dalton polymers orthe like) having a total molecular weight of any of the foregoing canalso be used. In one or more particular embodiments, depending upon theother features of the subject GLP-1 polymer conjugate, the conjugate isone that does not have one or more attached PEG moieties having aweight-average molecular weight of less than about 6,000 Daltons.

In instances in which the water-soluble polymer is a PEG, the PEG willtypically comprise a number of (OCH₂CH₂) monomers [or (CH₂CH₂O)monomers, depending on how the PEG is defined]. As used herein, thenumber of repeat units is typically identified by the subscript “n” in,for example, “(OCH₂CH₂)_(n).” Thus, the value of (n) typically fallswithin one or more of the following ranges: from 2 to about 3400, fromabout 100 to about 2300, from about 100 to about 2270, from about 136 toabout 2050, from about 225 to about 1930, from about 450 to about 1930,from about 1200 to about 1930, from about 568 to about 2727, from about660 to about 2730, from about 795 to about 2730, from about 795 to about2730, from about 909 to about 2730, and from about 1,200 to about 1,900.Preferred ranges of n include from about 10 to about 700, and from about10 to about 1800. For any given polymer in which the molecular weight isknown, it is possible to determine the number of repeating units (i.e.,“n”) by dividing the total weight-average molecular weight of thepolymer by the molecular weight of the repeating monomer.

With regard to the molecular weight of the water-soluble polymer, in ormore particular embodiments of the invention, depending upon the otherfeatures of the particular GLP-1 conjugate, the conjugate comprises aGLP-1 moiety covalently attached to a water-soluble polymer having amolecular weight greater than about 2,000 Daltons.

A polymer for use in the invention may be end-capped, that is, a polymerhaving at least one terminus capped with a relatively inert group, suchas a lower alkoxy group (i.e., a C₁₋₆ alkoxy group) or a hydroxyl group.One frequently employed end-capped polymer is methoxy-PEG (commonlyreferred to as mPEG), wherein one terminus of the polymer is a methoxy(—OCH₃) group. The —PEG-symbol used in the foregoing generallyrepresents the following structural unit:—CH₂CH₂O—(CH₂CH₂O)_(n)—CH₂CH₂—, where (n) generally ranges from aboutzero to about 4,000.

Multi-armed or branched PEG molecules, such as those described in U.S.Pat. No. 5,932,462, are also suitable for use in the present invention.For example, the PEG may be described generally according to thestructure: where poly_(a) and poly_(b) are PEG backbones (either thesame or different), such as methoxy poly(ethylene glycol); R″ is anon-reactive moiety, such as H, methyl or a PEG backbone; and P and Qare non-reactive linkages. In one embodiment, the branched PEG moleculeis one that includes a lysine residue, such as the following reactivePEG suitable for use in forming a GLP-1 conjugate. See, e.g, theShearwater Corporation Catalog, 2001, page 6. Although the branched PEGbelow is shown with a reactive succinimidyl group, this represents onlyone of a myriad of reactive functional groups suitable for reacting witha GLP-1 moiety.

In some instances, the polymeric reagent (as well as the correspondingconjugate prepared from the polymeric reagent) may lack a lysine residuein which the polymeric portions are connected to amine groups of thelysine via a “—OCH₂CONHCH₂CO—” group. In still other instances, thepolymeric reagent (as well as the corresponding conjugate prepared fromthe polymeric reagent) may lack a branched water-soluble polymer thatincludes a lysine residue (wherein the lysine residue is used to effectbranching).

Additional branched PEGs for use in forming a GLP-1 conjugate of thepresent invention include those described in co-owned U.S. PatentApplication Publication No. 2005/0009988. Representative branchedpolymers described therein include those having the followinggeneralized structure:

where POLY¹ is a water-soluble polymer; POLY² is a water-solublepolymer; (a) is 0, 1, 2 or 3; (b) is 0, 1, 2 or 3; (e) is 0, 1, 2 or 3;(f′) is 0, 1, 2 or 3; (g′) is 0, 1, 2 or 3; (h) is 0, 1, 2 or 3; (j) is0 to 20; each R¹ is independently H or an organic radical selected fromalkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, aryl and substituted aryl; X¹, when present, is aspacer moiety; X², when present, is a spacer moiety; X⁵, when present,is a spacer moiety; X⁶, when present, is a spacer moiety; X⁷, whenpresent, is a spacer moiety; X⁸, when present, is a spacer moiety; R⁵ isa branching moiety; and Z is a reactive group for coupling to a GLP-1moiety, optionally via an intervening spacer. POLY¹ and POLY² in thepreceding branched polymer structure may be different or identical,i.e., are of the same polymer type (structure) and molecular weight.

A preferred branched polymer falling into the above classificationsuitable for use in the present invention is:

where (m) is 2 to 4000, and (f) is 0 to 6 and (n) is 0 to 20.

Branched polymers suitable for preparing a conjugate of the inventionalso include those represented more generally by the formulaR(POLY)_(y), where R is a central or core molecule from which extends 2or more POLY arms such as PEG. The variable y represents the number ofPOLY arms, where each of the polymer arms can independently beend-capped or alternatively, possess a reactive functional group at itsterminus. A more explicit structure in accordance with this embodimentof the invention possesses the structure, R(POLY-Z)_(y), where each Z isindependently an end-capping group or a reactive group, e.g., suitablefor reaction with a GLP-1 moiety. In yet a further embodiment when Z isa reactive group, upon reaction with a GLP-1 moiety, the resultinglinkage can be hydrolytically stable, or alternatively, may bedegradable, i.e., hydrolyzable. Typically, at least one polymer armpossesses a terminal functional group suitable for reaction with, e.g.,a GLP-1 moiety. Branched PEGs such as those represented generally by theformula, R(PEG)_(y) above possess 2 polymer arms to about 300 polymerarms (i.e., n ranges from 2 to about 300). Preferably, such branchedPEGs typically possess from 2 to about 25 polymer arms, such as from 2to about 20 polymer arms, from 2 to about 15 polymer arms, or from 3 toabout 15 polymer arms. Multi-armed polymers include those having 3, 4,5, 6, 7 or 8 arms.

Core molecules in branched PEGs as described above include polyols,which are then further functionalized. Such polyols include aliphaticpolyols having from 1 to 10 carbon atoms and from 1 to 10 hydroxylgroups, including ethylene glycol, alkane diols, alkyl glycols,alkylidene alkyl diols, alkyl cycloalkane diols, 1,5-decalindiol,4,8-bis(hydroxymethyl)tricyclodecane, cycloalkylidene diols,dihydroxyalkanes, trihydroxyalkanes, and the like. Cycloaliphaticpolyols may also be employed, including straight chained or closed-ringsugars and sugar alcohols, such as mannitol, sorbitol, inositol,xylitol, quebrachitol, threitol, arabitol, erythritol, adonitol,ducitol, facose, ribose, arabinose, xylose, lyxose, rhamnose, galactose,glucose, fructose, sorbose, mannose, pyranose, altrose, talose,tagitose, pyranosides, sucrose, lactose, maltose, and the like.Additional aliphatic polyols include derivatives of glyceraldehyde,glucose, ribose, mannose, galactose, and related stereoisomers. Othercore polyols that may be used include crown ether, cyclodextrins,dextrins and other carbohydrates such as starches and amylose. Typicalpolyols include glycerol, pentaerythritol, sorbitol, andtrimethylolpropane.

As will be described in more detail in the linker section below,although any of a number of linkages can be used to covalently attach apolymer to a GLP-1 moiety, in certain instances, the linkage isdegradable, designated herein as L_(D), that is to say, contains atleast one bond or moiety that hydrolyzes under physiological conditions,e.g., an ester, hydrolyzable carbamate, carbonate, or other such group.

Illustrative multi-armed PEGs having 3 arms, 4-aims, and 8 armscorrespond to those in Nektar's Advanced PEGylation Catalog 2005-2006,page 26. Multi-armed activated polymers for use in the method of theinvention include those corresponding to the following structure, whereE represents a reactive group suitable for reaction with a reactivegroup on the GLP-1 moiety. In one or more embodiments, E is an —OH (forreaction with a GLP-1 carboxy group or equivalent), a carboxylic acid orequivalent (such as an active ester), a carbonic acid (for reaction withGLP-1-OH groups), or an amino group.

In the structure above, PEG is —(CH₂CH₂O)_(n)CH₂CH₂—, and m is selectedfrom 3, 4, 5, 6, 7, and 8. In certain embodiments, typical linkages areester, carboxyl and hydrolyzable carbamate, such that thepolymer-portion of the conjugate is hydrolyzed in vivo to release theGLP-1 moiety from the intact polymer conjugate. In such instances, thelinker L is designated as L_(D).

Alternatively, the polymer may possess an overall forked structure asdescribed in U.S. Pat. No. 6,362,254. This type of polymer segment isuseful for reaction with two GLP-1 moieties, where the two GLP-1moieties are positioned a precise or predetermined distance apart.

In any of the representative structures provided herein, one or moredegradable linkages may additionally be contained in the polymersegment, POLY, to allow generation in vivo of a conjugate having asmaller PEG chain than in the initially administered conjugate.Appropriate physiologically cleavable (i.e., releasable) linkagesinclude but are not limited to ester, carbonate ester, carbamate,sulfate, phosphate, acyloxyalkyl ether, acetal, and ketal. Such linkageswhen contained in a given polymer segment will often be stable uponstorage and upon initial administration.

The PEG polymer used to prepare a GLP-1 polymer conjugate may comprise apendant PEG molecule having reactive groups, such as carboxyl or amino,covalently attached along the length of the PEG rather than at the endof the PEG chain(s). The pendant reactive groups can be attached to thePEG directly or through a spacer moiety, such as an alkylene group.

Additional representative PEGs having either linear or branchedstructures for use in forming a conjugate of the invention may bepurchased from Nektar Therapeutics (formerly Shearwater Corporation,Huntsville, Ala.). Illustrative PEG reagents are described in Nektar's2005-2006 catalogue entitled, “Polyethylene Glycol and Derivatives forAdvanced PEGylation,” the contents of which is expressly incorporatedherein by reference.

Releasable Linkage

Preferably, a GLP-1 polymer conjugate according to one aspect of theinvention is one comprising a GLP-1 moiety releasably attached,preferably at its N-terminus, to a water-soluble polymer. Hydrolyticallydegradable linkages, useful not only as a degradable linkage within apolymer backbone, but also, in the case of certain preferred embodimentsof the invention, for covalently attaching a water-soluble polymer to aGLP-1 moiety, include: carbonate; imine resulting, for example, fromreaction of an amine and an aldehyde (see, e.g., Ouchi et al. (1997)Polymer Preprints 38(1):582-3); phosphate ester, formed, for example, byreacting an alcohol with a phosphate group; hydrazone, e.g., formed byreaction of a hydrazide and an aldehyde; acetal, e.g., formed byreaction of an aldehyde and an alcohol; orthoester, formed, for example,by reaction between a formate and an alcohol; and esters, and certainurethane (carbamate) linkages.

Illustrative PEG reagents for use in preparing a releasable GLP-1conjugate in accordance with the invention are described in U.S. Pat.Nos. 6,348,558, 5,612,460, 5,840,900, 5,880,131, and 6,376,470.

Additional PEG reagents for use in the invention include hydrolyzable orreleasable PEGs and linkers such as those described in U.S. patentapplication Ser. No. 11/454,971, filed Jun. 16, 2006. In the resultingconjugate, the GLP-1 moiety and the polymer are each covalently attachedto different positions of the scaffold aromatic, e.g., Fmoc or FMS,structure, and are releasable under physiological conditions.Generalized structures corresponding to the polymers described thereinare provided below.

For example, one such polymeric reagent comprises the followingstructure:

where POLY¹ is a first water-soluble polymer; POLY² is a secondwater-soluble polymer; X¹ is a first spacer moiety; X² is a secondspacer moiety;

is an aromatic-containing moiety bearing an ionizable hydrogen atom,H_(□); R¹ is H or an organic radical; R² is H or an organic radical; and(FG) is a functional group capable of reacting with an amino group of anactive agent to form a degradable linkage, such as a carbamate linkage(such as N-succinimidyloxy, 1-benzotriazolyloxy, oxycarbonylimidazole,—O—C(O)—Cl, O—C(O)—Br, unsubstituted aromatic carbonate radicals andsubstituted aromatic carbonate radicals). The polymeric reagent caninclude one, two, three, four or more electron altering groups attachedto the aromatic-containing moiety.

Preferred aromatic-containing moieties are bicyclic and tricyclicaromatic hydrocarbons. Fused bicyclic and tricyclic aromatics includepentalene, indene, naphthalene, azulene, heptalene, biphenylene,as-indacene, s-indacene, acenaphthylene, fluorene, phenalene,phenanthrene, anthracene, and fluoranthene.

A preferred polymer reagent possesses the following structure,

where mPEG corresponds to CH₃O—(CH₂CH₂O)_(n)CH₂CH₂—, X₁ and X₁ are eachindependently a spacer moiety having an atom length of from about 1 toabout 18 atoms, n ranges from 10 to 1800, p is an integer ranging from 1to 8, R₁ is H or lower alkyl, R₂ is H or lower alkyl, and Ar is anaromatic hydrodrocarbon, preferably a bicyclic or tricyclic aromatichydrocarbon. FG is as defined above. Preferably, FG corresponds to anactivated carbonate ester suitable for reaction with an amino group onGLP-1. Preferred spacer moieties, X₁ and X₂, include —NH—C(O)—CH₂—O—,—NH—C(O)—(CH₂)_(q)—O—, —NH—C(O)—(CH₂)_(q)—C(O)—NH—, —NH—C(O)—(CH₂)_(q)—,and —C(O)—NH—, where q is selected from 2, 3, 4, and 5. Preferably,although not necessarily, the nitrogen in the preceding spacers isproximal to the PEG rather than to the aromatic moiety.

Another such branched (2-armed) polymeric reagent comprised of twoelectron altering groups comprises the following structure:

wherein each of POLY¹, POLY², X¹, X², R¹, R²,

and (FG) is as defined immediately above, and R^(e1) is a first electronaltering group; and R^(e2) is a second electron altering group. Anelectron altering group is a group that is either electron donating (andtherefore referred to as an “electron donating group”), or electronwithdrawing (and therefore referred to as an “electron withdrawinggroup”). When attached to the aromatic-containing moiety bearing anionizable hydrogen atom, an electron donating group is a group havingthe ability to position electrons away from itself and closer to orwithin the aromatic-containing moiety. When attached to thearomatic-containing moiety bearing an ionizable hydrogen atom, anelectron withdrawing group is a group having the ability to positionelectrons toward itself and away from the aromatic-containing moiety.Hydrogen is used as the standard for comparison in the determination ofwhether a given group positions electrons away or toward itself.Preferred electron altering groups include, but are not limited to,—CF₃, —CH₂CF₃, —CH₂C₆F₅, —CN, —NO₂, —S(O)R, —S(O)Aryl, —S(O₂)R,—S(O₂)Aryl, —S(O₂)OR, —S(O₂)OAryl, —S(O₂)NHR, —S(O₂)NHAryl, —C(O)R,—C(O)Aryl, —C(O)OR, —C(O)NHR, and the like, wherein R is H or an organicradical.

An additional branched polymeric reagent suitable for use in the presentinvention comprises the following structure:

where POLY¹ is a first water-soluble polymer; POLY² is a secondwater-soluble polymer; X¹ is a first spacer moiety; X² is a secondspacer moiety; Ar¹ is a first aromatic moiety; Ar¹ is a second aromaticmoiety; H_(α) is an ionizable hydrogen atom; R¹ is H or an organicradical; R² is H or an organic radical; and (FG) is a functional groupcapable of reacting with an amino group of GLP-1 to form a degradablelinkage, such as carbamate linkage.

Another exemplary polymeric reagent comprises the following structure:

wherein each of POLY¹, POLY², X¹, X², Ar¹, Ar², H_(α), R¹, R², and (FG)is as previously defined, and R^(e1) is a first electron altering group.While stereochemistry is not specifically shown in any structureprovided herein, the provided structures contemplate both enantiomers,as well as compositions comprising mixtures of each enantiomer in equalamounts (i.e., a racemic mixture) and unequal amounts.

Yet an additional polymeric reagent for use in preparing a GLP-1conjugate possesses the following structure:

wherein each of POLY¹, POLY², X¹, X², Ar¹, Ar², H_(α), R¹, R², and (FG)is as previously defined, and R^(e1) is a first electron altering group;and R^(e2) is a second electron altering group.

A preferred polymeric reagent comprises the following structure:

wherein each of POLY¹, POLY², X¹, X², R¹, R², H_(α) and (FG) is aspreviously defined, and, as can be seen from the structure above, thearomatic moiety is a fluorene. The POLY arms substituted on the fluorenecan be in any position in each of their respective phenyl rings, i.e.,POLY¹-X¹— can be positioned at any one of carbons 1, 2, 3, and 4, andPOLY²—X²— can be in any one of positions 5, 6, 7, and 8.

Yet another preferred fluorene-based polymeric reagent comprises thefollowing structure:

wherein each of POLY¹, POLY², X¹, X², R¹, R², H_(α) and (FG) is aspreviously defined, and R^(e1) is a first electron altering group; andR^(e2) is a second electron altering group as described above.

Yet another exemplary polymeric reagent for conjugating to a GLP-1moiety comprises the following fluorene-based structure:

wherein each of POLY¹, POLY², X¹, X², R¹, R², H_(α) and (FG) is aspreviously defined, and R^(e1) is a first electron altering group; andR^(e2) is a second electron altering group.

Particular fluorene-based polymer reagents for forming a releasableGLP-1 polymer conjugate in accordance with the invention include thefollowing:

Still another exemplary polymeric reagent comprises the followingstructure:

wherein each of POLY¹, POLY², X¹, X², R¹, R², H_(α) and (FG) is aspreviously defined, and R^(e1) is a first electron altering group; andR^(e2) is a second electron altering group. The syntheses and chemicalstructures of preferred branched releasable polymer reagents containinga fluorene scaffold, as well as their covalent attachment to GLP-1 areprovided in Examples 1, 2, 3, 4, 5, 6, and 24. Such branched reagentssuitable for preparing a releasable GLP-1 conjugate includeN-{di(mPEG(20,000)oxymethylcarbonylamino)fluoren-9-ylmethoxycarbonyloxy}succinimide,N-[2,7 di(4 mPEG(10,000)aminocarbonylbutyrylamino)fluoren-9ylmethoxycarbonyloxy]-succinimide (“G2PEG2Fmoc_(20k)-NHS”), andPEG2-CAC-Fmoc_(4k)-BTC (Example 24). Of course, PEGs of any molecularweight as set forth herein may be employed in the above structures, andthe particular activating groups described above are not meant to belimiting in any respect, and may be substituted by any other suitableactivating group suitable for reaction with a reactive group present onthe GLP-1 moiety.

Those of ordinary skill in the art will recognize that the foregoingdiscussion describing water-soluble polymers for use in forming a GLP-1conjugate is by no means exhaustive and is merely illustrative, and thatall polymeric materials having the qualities described above arecontemplated. As used herein, the term “polymeric reagent” generallyrefers to an entire molecule, which can comprise a water-soluble polymersegment, as well as additional spacers and functional groups.

GLP-1 Conjugates

As described above, a conjugate of the invention comprises awater-soluble polymer covalently attached (either directly or through aspacer or linker moiety) to a GLP-1 moiety. Typically, for any givenconjugate, there will be one to four water-soluble polymers covalentlyattached to a GLP-1 moiety (wherein for each water-soluble polymer, thewater-soluble polymer can be attached either directly to the GLP-1moiety or through a spacer moiety).

That is to say, a GLP-1 conjugate of the invention typically has 1, 2,3, or 4 water-soluble polymers individually attached to a GLP-1 moiety.That is to say, in certain embodiments, a conjugate of the inventionwill possess not more than 4 water-soluble polymers individuallyattached to a GLP-1 moiety, or not more than 3 water-soluble polymersindividually attached to a GLP-1 moiety, or not more than 2water-soluble polymers individually attached to a GLP-1 moiety, or notmore than 1 water-soluble polymer attached to a GLP-1 moiety.Preferably, the structure of each of the water-soluble polymers attachedto the GLP-1 moiety is the same. One particularly preferred GLP-1conjugate in accordance with the invention is one having a water-solublepolymer releasably attached to the N-terminus of GLP-1. Additionalwater-soluble polymers may be releasably attached to other sites on theGLP-1 moiety, e.g., such as one or two additional sites. For example, aGLP-1 conjugate having a water-soluble polymer releasably attached tothe N-terminus may additionally possess a water-soluble polymerreleasably attached Lys26, and/or to Lys34. Another particular preferredconjugate of the present invention is a mono-GLP-1 polymer conjugate,i.e., a GLP-1 moiety having one water-soluble polymer covalentlyattached thereto. Even more preferably, the water-soluble polymer is onethat is releasably attached to the GLP-1 moiety at its N-terminus.

Preferably, a GLP-1 polymer conjugate of the invention is absent a metalion, i.e., the GLP-1 moiety is not chelated to a metal ion.

For the GLP-1 polymer conjugates described herein, the GLP-1 moiety mayoptionally possess one or more N-methyl substituents, e.g., at any oneor more of positions 7-His, 8-Ala, and 9-Glutamic Acid. Alternatively,for the GLP-1 polymer conjugates described herein, the GLP-1 moiety maybe glycosylated, e.g., having a mono- or disaccharide covalentlydescribed to one or more sites thereof. Particularly preferredglycosylation sites include the N-terminus, Ala8, Glu9, Thr11, Thr13,Ser14, Ser17, Ser18, Glu21, Gly22, Gln23, Lys26, and Lys34.

As discussed herein, the compounds of the present invention may be madeby any of the various methods and techniques known and available tothose skilled in the art.

The Linkage

The particular linkage between the GLP-1 moiety and the water-solublepolymer (or the spacer moiety that is attached to the water-solublepolymer) depends on a number of factors. Such factors include, forexample, the particular linkage chemistry employed, the particular GLP-1moiety, the available functional groups within the GLP-1 moiety (eitherfor attachment to a polymer or conversion to a suitable attachmentsite), the possible presence of additional reactive functional groupswithin the GLP-1 moiety due to methylation and/or glycosylation, and thelike.

In one or more embodiments of the invention, the linkage between theGLP-1 moiety and the water-soluble polymer is a releasable linkage. Thatis, the water-soluble polymer is cleaved (either through hydrolysis, anenzymatic processes, or otherwise), thereby resulting in the native oran unconjugated GLP-1 moiety. Preferably, the releasable linkage is ahydrolytically degradable linkage, where upon hydrolysis, the nativeGLP-1 moiety, or a slightly modified version thereof, is released. Thereleasable linkage may result in the water-soluble polymer (and anyspacer moiety) detaching from the GLP-1 moiety in vivo (and in vitro)without leaving any fragment of the water-soluble polymer (and/or anyspacer or linker moiety) attached to the GLP-1 moiety. Exemplaryreleasable linkages include carbonate, carboxylate ester, phosphateester, thiolester, anhydrides, acetals, ketals, acyloxyalkyl ether,imines, carbamates, and orthoesters. Such linkages can be readily formedby reaction of the GLP-1 moiety and/or the polymeric reagent usingcoupling methods commonly employed in the art. Hydrolyzable linkages areoften readily formed by reaction of a suitably activated polymer with anon-modified functional group contained within the GLP-1 moiety.Preferred positions for covalent attachment of a water-soluble polymerinduce the N-terminal, the C-terminal, as well as the internal lysines.Preferred releasable linkages include carbamate and ester.

Generally speaking, a preferred GLP-1 conjugate of the invention willpossess the following generalized structure:

where POLY is a water-soluble polymer such as any of the illustrativepolymer reagents provided in the tables herein, L_(D) is a hydrolyzablelinkage, and k is an integer selected from 1, 2, and 3. In thegeneralized structure above, L_(D) refers to the hydrolyzable linkageper se (e.g., a carbamate or an ester linkage), while “POLY” is meant toinclude the polymer repeat units, e.g., CH₃(OCH₂CH₂)_(n), as well as anyadditional linker or spacer atoms interposed between the polymer repeatunits and the hydrolyzable linkage. In a preferred embodiment of theinvention, at least one of the water-soluble polymer molecules iscovalently attached to the N-terminus of GLP-1. In one embodiment of theinvention, k equals 1 and L_(D) is —O—C(O)—NH—, where the —NH— is partof the GLP-1 residue and represents an amino group thereof.

Although releasable linkages are preferred, the linkage between theGLP-1 moiety and the water-soluble polymer (or the linker moiety that isattached to the polymer) may be a hydrolytically stable linkage, such asan amide, a urethane (also known as carbamate), amine, thioether (alsoknown as sulfide), or urea (also known as carbamide). One suchembodiment of the invention comprises GLP-1 having a water-solublepolymer such as PEG covalently attached at the N-terminus of GLP-1. Insuch instances, alkylation of the N-terminal residue permits retentionof the charge on the N-terminal nitrogen.

With regard to linkages, in one more embodiments of the invention, aconjugate is provided that comprises a GLP-1 moiety covalently attachedat an amino acid residue, either directly or through a linker comprisedof one or more atoms, to a water-soluble polymer.

The conjugates (as opposed to an unconjugated GLP-1 moiety) may or maynot possess a measurable degree of GLP-1 activity. That is to say, aconjugate in accordance with the invention will typically possessanywhere from about 0% to about 100% or more of the bioactivity of theunmodified parent GLP-1 moiety. Typically, compounds possessing littleor no GLP-1 activity contain a releasable linkage connecting the polymerto the GLP-1 moiety, so that regardless of the lack of activity in theconjugate, the active parent molecule (or a derivative thereof havingGLP-1 activity) is released by degradation of the linkage (e.g.,hydrolysis upon aqueous-induced cleavage of the linkage). Such activitymay be determined using a suitable in vivo or in vitro model, dependingupon the known activity of the particular moiety having GLP-1 activityemployed.

Optimally, degradation of a linkage is facilitated through the use ofhydrolytically cleavable and/or enzymatically degradable linkages suchas urethane, amide, certain carbamate, carbonate or ester-containinglinkages. In this way, clearance of the conjugate via cleavage ofindividual water-soluble polymer(s) can be modulated by selecting thepolymer molecular size and the type of functional group for providingthe desired clearance properties. In certain instances, a mixture ofpolymer conjugates is employed where the polymers possess structural orother differences effective to alter the release (hydrolysis rate) ofthe GLP-1 moiety, such that one can achieve a desired sustained deliveryprofile.

One of ordinary skill in the art can determine the proper molecular sizeof the polymer as well as the cleavable functional group, depending uponseveral factors including the mode of administration. For example, oneof ordinary skill in the art, using routine experimentation, candetermine a proper molecular size and cleavable functional group byfirst preparing a variety of polymer-(GLP-1) conjugates with differentweight-average molecular weights, degradable functional groups, andchemical structures, and then obtaining the clearance profile for eachconjugate by administering the conjugate to a patient and takingperiodic blood and/or urine samples. Once a series of clearance profileshas been obtained for each tested conjugate, a conjugate or mixture ofconjugates having the desired clearance profile(s) can be determined.

For conjugates possessing a hydrolytically stable linkage that couplesthe GLP-1 moiety to the water-soluble polymer, the conjugate willtypically possess a measurable degree of GLP-1 activity. For instance,such conjugates are typically characterized as having a bioactivitysatisfying one or more of the following percentages relative to that ofthe unconjugated GLP-1 moiety: at least about 2%, at least about 5%, atleast about 10%, at least about 15%, at least about 25%, at least about30%, at least about 40%, at least about 50%, at least about 60%, atleast about 80%, at least about 85%, at least about 90%, at least about95%, at least about 97%, at least about 100%, and more than 105% (whenmeasured in a suitable model, such as those presented here and/or knownin the art). Often, conjugates having a hydrolytically stable linkage(e.g., an amide linkage) will possess at least some degree of thebioactivity of the unmodified parent GLP-1 moiety. Due to their extendedhalf-lives,

Exemplary conjugates in accordance with the invention will now bedescribed.

The GLP-1 moiety is expected to share (at least in part) an amino acidsequence similar or related to a human GLP-1. Thus, as previouslyindicated, while reference will be made to specific locations or atomswithin a GLP-1 sequence, such a reference is for convenience only andone having ordinary skill in the art will be able to readily determinethe corresponding location or atom in other moieties having GLP-1activity. In particular, the description provided herein for a humanGLP-1 is also often applicable not only to a human GLP-1, but tofragments, deletion variants, substation variants and addition variantsof any of the foregoing.

Amino groups on a GLP-1 moiety provide a point of attachment between theGLP-1 moiety and the water-soluble polymer. For example, SEQ ID NOS. 1and 2 each comprise two lysine residues, each lysine residue containingan ε-amino group that may be available for conjugation, as well as oneamino terminus. See SEQ ID NO: 1 and SEQ ID NO: 2. Thus, exemplaryattachment points include attachment at an amino acid (through theamine-containing side chain of a lysine residue) at either or both ofpositions 26 and 34. Typical attachment points of GLP-1 includeattachment at any one of positions 26, 34, and the N-terminus. In one ormore embodiments, attachment is a single attachment at one of positions26, 34 or the N-terminus (His 7).

There are a number of examples of suitable water-soluble polymericreagents useful for forming covalent linkages with available amines of aGLP-1 moiety. Certain specific examples, along with the correspondingconjugates, are provided in Table 1 below. In the table, the variable(n) represents the number of repeating monomeric units and “(GLP-1)”represents a GLP-1 moiety following conjugation to the water-solublepolymer. While each polymeric portion [e.g., (OCH₂CH₂)_(n) or(CH₂CH₂O)_(n)] presented in Table 1 terminates in a “CH₃” group, othergroups (such as H and benzyl) can be substituted therefore.

As will be clearly understood by one skilled in the art, for conjugatessuch as those set forth below resulting from reaction with a GLP-1 aminogroup, the amino group extending from the GLP-1 designation “˜NH-GLP-1”represents the residue of the GLP-1 moiety itself in which the ˜NH— isan amino group of the GLP-1 moiety. One preferred site of attachment forthe polymer reagents shown below is the N-terminus. Further, althoughthe conjugates in the Tables herein illustrate a single water-solublepolymer covalently attached to a GLP-1 moiety, it will be understoodthat the conjugate structures on the right are meant to also encompassconjugates having more than one of such water-soluble polymer moleculescovalently attached to GLP-1, e.g., 2, 3, or 4 water-soluble polymermolecules.

TABLE 1 Amine-Specific Polymeric Reagents and the GLP-1 Moiety ConjugateFormed Therefrom Polymeric Reagent Corresponding Conjugate

  mPEG-Oxycarbonylimidazole Reagent

    Carbamate Linkage

  mPEG Nitrophenyl Reagent

    Carbamate Linkage

  mPEG-Trichlorophenyl Carbonate Reagent

            Carbamate Linkage

  Fmoc-NHS Reagent

    Carbamate Linkage

  Fmoc-NHS Reagent

    Carbamate Linkage

  Fmoc-NHS Reagent

    Carbamate Linkage

  Fmoc-BTC Reagent

    Carbamate Linkage

  mPEG-Succinimidyl Reagent

          Amide Linkage

  Homobifunctional PEG-Succinimidyl Reagent

          Amide Linkages

  Heterobifunctional PEG-Succinimidyl Reagent

      Amide Linkage

  mPEG-Succinimidyl Reagent

          Amide Linkage

  mPEG-Succinimdyl Reagent

          Amide Linkage

  mPEG-Succinimidyl Reagent

          Amide Linkage

  mPEG-Succinimidyl Reagent

          Amide Linkage

  mPEG-Benzotriazole Carbonate Reagent

          Carbamate Linkage

  mPEG-Succinimidyl Reagent

        Carbamate Linkage

  mPEG-Succinimidyl Reagent

        Amide Linkage

  mPEG Succinimidyl Reagent

          Amide Linkage

  Branched mPEG2-N-Hydroxysuccinimide Reagent

  Amide Linkage

  Branched mPEG2-Aldehyde Reagent

  Secondary Amine Linkage

  mPEG-Succinimidyl Reagent

      Amide Linkage

  mPEG-Succinimidyl Reagent

          Amide Linkage

  Homobifunctional PEG-Succinimidyl Reagent

      Amide Linkages

  mPEG-Succinimidyl Reagent

      Amide Linkage

  Homobifunctional PEG-Succinimidyl Propionate Reagent

      Amide Linkages

  mPEG-Succinimidyl Reagent

      Amide Linkage

  Branched mPEG2-N-Hydroxysuccinimide Reagent

    Amide Linkage

  Branched mPEG2-N-Hydroxysuccinimide Reagent

    Amide Linkage

  mPEG-Thioester Reagent

    Amide Linkage

  Homobifunctional PEG Propionaldehyde Reagent

  Secondary Amine Linkages

  mPEG Propionaldehyde Reagent

      Secondary Amine Linkage

  Homobifunctional PEG Butyraldehyde Reagent

  Secondary Amine Linkages

  mPEG Butryaldehyde Reagent

      Secondary Amine Linkage

      mPEG Butryaldehyde Reagent

  Secondary Amine Linkage

  Homobifunctional PEG Butryaldehyde Reagent

    Secondary Amine Linkages

  Branched mPEG2 Butyraldehyde Reagent

  Secondary Amine Linkage

  Branched mPEG2 Butyraldehyde Reagent

      Secondary Amine Linkage

  mPEG Acetal Reagent

      Secondary Amine Linkage

  mPEG Piperidone Reagent

  Secondary Amine Linkage (to a secondary carbon)

  mPEG Methylketone Reagent

  secondary amine linkage (to a secondary carbon)

  mPEG Tresylate Reagent

        Secondary Amine Linkage

  mPEG Maleimide Reagent (under certain reaction conditions such as pH >8)

    Secondary Amine Linkage

  mPEG Maleimide Reagent (under certain reaction conditions such as pH >8)

    Secondary Amine Linkage

        mPEG Maleimide Reagent (under certain reaction conditions suchas pH > 8)

  Secondary Amine Linkage

  mPEG Forked Maleimide Reagent (under certain reaction conditions suchas pH > 8)

    Secondary Amine Linkages

  Branched mPEG2 Maleimide Reagent (under certain reaction conditionssuch as pH > 8)

    Secondary Amine Linkage

Amine Conjugation and Resulting Conjugates

Conjugation of a polymeric reagent to an amine group of a GLP-1 moietycan be accomplished by a variety of techniques. In one approach, a GLP-1moiety is conjugated to a polymeric reagent functionalized with anactive ester such as a succinimidyl derivative (e.g., anN-hydroxysuccinimide ester). In this approach, the polymeric reagentbearing the reactive ester is reacted with the GLP-1 moiety in aqueousmedia under appropriate pH conditions, e.g., from pHs ranging from about3 to about 8, about 3 to about 7, or about 4 to about 6.5. Most polymeractive esters can couple to a target protein such as GLP-1 atphysiological pH, e.g., at 7.0. However, less reactive derivatives mayrequire a higher pH. Typically, activated PEGs can be attached to aprotein such as GLP-1 at pHs from about 7.0 to about 10.0 for covalentattachment to an internal lysine. Typically, lower pHs are used, e.g., 4to about 5.75, for preferential covalent attachment to the N-terminus.Thus, different reaction conditions (e.g., different pHs or differenttemperatures) can result in the attachment of a water-soluble polymersuch as PEG to different locations on the GLP-1 moiety (e.g., internallysines versus the N-terminus). Coupling reactions can often be carriedout at room temperature, although lower temperatures may be required forparticularly labile GLP-1 moieties. Reaction times are typically on theorder of minutes, e.g., 30 minutes, to hours, e.g., from about 1 toabout 36 hours), depending upon the pH and temperature of the reaction.N-terminal PEGylation, e.g., with a PEG reagent bearing an aldehydegroup, is typically conducted under mild conditions, pHs from about5-10, for about 6 to 36 hours. Varying ratios of polymer reagent toGLP-1 moiety may be employed, e.g., from an equimolar ratio up to a10-fold molar excess of polymer reagent. Typically, up to a 5-fold molarexcess of polymer reagent will suffice.

In certain instances, it may be preferable to protect certain aminoacids from reaction with a particular polymer reagent if site specificcovalent attachment is desired using commonly employedprotection/deprotection methodologies such as those well known in theart. Mono-GLP-1 conjugates selectively conjugated at the N-terminus ofGLP-1 were prepared as described in detail in the Examples hereinwithout the need to employ protection/deprotection strategies.

In an alternative approach to direct coupling reactions, the PEG reagentmay be incorporated at a desired position of the GLP-1 moiety duringpeptide synthesis. In this way, site-selective introduction of one ormore PEGs can be achieved. See, e.g., International Patent PublicationNo. WO 95/00162, which describes the site selective synthesis ofconjugated peptides.

Exemplary conjugates that can be prepared using, for example, polymericreagents containing a reactive ester for coupling to an amino group ofGLP-1, comprise the following alpha-branched structure:

where POLY is a water-soluble polymer, (a) is either zero or one; X¹,when present, is a spacer moiety comprised of one or more atoms; R¹ ishydrogen an organic radical; and “˜NH-GLP-1” represents a residue of aGLP-1 moiety, where the underlined amino group represents an amino groupof the GLP-1 moiety.

With respect to the structure corresponding to that referred to in theimmediately preceding paragraph, any of the water-soluble polymersprovided herein can be defined as POLY, any of the spacer moietiesprovided herein can be defined as X¹ (when present), any of the organicradicals provided herein can be defined as R¹ (in instances where R¹ isnot hydrogen), and any of the GLP-1 moieties provided herein can bedefined as GLP-1. In one or more embodiments corresponding to thestructure referred to in the immediately preceding paragraph, POLY is apoly(ethylene glycol) such as H₃CO(CH₂CH₂O)_(n)—, wherein (n) is aninteger having a value of from 3 to 4000, more preferably from 10 toabout 1800; (a) is one; X¹ is a C₁₋₆ alkylene, such as one selected frommethylene (i.e., —CH₂—), ethylene (i.e., —CH₂—CH₂—) and propylene (i.e.,—CH₂—CH₂—CH₂—); R¹ is H or lower alkyl such as methyl or ethyl; andGLP-1 corresponds to SEQ ID NO:1 or SEQ ID NO:2.

Typical of another approach for conjugating a GLP-1 moiety to apolymeric reagent is reductive amination. Typically, reductive aminationis employed to conjugate a primary amine of a GLP-1 moiety with apolymeric reagent functionalized with a ketone, aldehyde or a hydratedform thereof (e.g., ketone hydrate and aldehyde hydrate). In thisapproach, the primary amine from the GLP-1 moiety (e.g., the N-terminus)reacts with the carbonyl group of the aldehyde or ketone (or thecorresponding hydroxy-containing group of a hydrated aldehyde orketone), thereby forming a Schiff base. The Schiff base, in turn, isthen reductively converted to a stable conjugate through use of areducing agent such as sodium borohydride or any other suitable reducingagent. Selective reactions (e.g., at the N-terminus are possible) arepossible, particularly with a polymer functionalized with a ketone or analpha-methyl branched aldehyde and/or under specific reaction conditions(e.g., reduced pH).

Exemplary conjugates that can be prepared using, for example, polymericreagents containing an aldehyde (or aldehyde hydrate) or ketone or(ketone hydrate) possess the following structure:

where POLY is a water-soluble polymer; (d) is either zero or one; X²,when present, is a spacer moiety comprised of one or more atoms; (b) isan integer having a value of one through ten; (c) is an integer having avalue of one through ten; R², in each occurrence, is independently H oran organic radical; R³, in each occurrence, is independently H or anorganic radical; and “˜NH-GLP-1” represents a residue of a GLP-1 moiety,where the underlined amino group represents an amino group of the GLP-1moiety.

Yet another illustrative conjugate of the invention possesses thestructure:

where k ranges from 1 to 3, and n ranges from 10 to about 1800.

With respect to the structure corresponding to that referred to inimmediately preceding paragraph, any of the water-soluble polymersprovided herein can be defined as POLY, any of the spacer moietiesprovided herein can be defined as X² (when present), any of the organicradicals provided herein can be independently defined as R² and R³ (ininstances where R² and R³ are independently not hydrogen), and any ofthe GLP-1 moieties provided herein can be defined as GLP-1. In one ormore embodiments of the structure referred to in the immediatelypreceding paragraph, POLY is a poly(ethylene glycol) such asH₃CO(CH₂CH₂O)_(n)—, wherein (n) is an integer having a value of from 3to 4000, more preferably from 10 to about 1800; (d) is one; X¹ is amide[e.g., —C(O)NH-]; (b) is 2 through 6, such as 4; (c) is 2 through 6,such as 4; each of R² and R³ are independently H or lower alkyl, such asmethyl when lower alkyl; and GLP-1 is human GLP-1.

Another example of a GLP-1 conjugate in accordance with the inventionhas the following structure:

wherein each (n) is independently an integer having a value of from 3 to4000, preferably from 10 to 1800; X² is as previously defined; (b) is 2through 6; (c) is 2 through 6; R², in each occurrence, is independentlyH or lower alkyl; and “˜NH-GLP-1” represents a residue of a GLP-1moiety, where the underlined amino group represents an amino group ofthe GLP-1 moiety.

Additional GLP-1 polymer conjugates resulting from reaction of awater-soluble polymer with an amino group of GLP-1 are provided below.The following conjugate structures are releasable. One such structurecorresponds to:

where mPEG is CH₃O—(CH₂CH₂O)_(n)CH₂CH₂—, n ranges from 10 to 1800, p isan integer ranging from 1 to 8, R₁ is H or lower alkyl, R₂ is H or loweralkyl, Ar is an aromatic hydrocarbon, such as a fused bicyclic ortricyclic aromatic hydrocarbon, X₁ and X₂ are each independently aspacer moiety having an atom length of from about 1 to about 18 atoms,˜NH-GLP-1 is as previously described, and k is an integer selected from1, 2, and 3. The value of k indicates the number of water-solublepolymer molecules attached to different sites on the GLP-1 moiety. In apreferred embodiment, R₁ and R₂ are both H. The spacer moieties, X₁ andX₂, preferably each contain one amide bond; In a preferred embodiment,X₁ and X₂ are the same. Preferred spacers, i.e., X₁ and X₂, include—NH—C(O)—CH₂—O—, —NH—C(O)—(CH₂)_(q)—O—, —NH—C(O)—(CH₂)_(q)—C(O)—NH—,—NH—C(O)—(CH₂)_(q)—, and —C(O)—NH—, where q is selected from 2, 3, 4,and 5. Although the spacers can be in either orientation, preferably,the nitrogen is proximal to the PEG rather than to the aromatic moiety.Illustrative aromatic moieties include pentalene, indene, naphthalene,indacene, acenaphthylene, and fluorene.

Particularly preferred conjugates of this type are provided below.

Additional GLP-1 conjugates resulting from covalent attachment to aminogroups of GLP-1 that are also releasable include the following:

where L_(D1) is either —O— or —NH—C(O)—, Ar₁ is an aromatic group, e.g.,ortho, meta, or para-substituted phenyl, and k is an integer selectedfrom 1, 2, and 3. Particular conjugates of this type include:

where n ranges from about 10 to about 1800.

Additional releasable conjugates in accordance with the invention areprepared using water-soluble polymer reagents such as those described inU.S. Pat. No. 6,214,966. Such water-soluble polymers are degradable, andpossess at least one hydrolytically degradable ester linkage close tothe covalent attachment to the active agent. The polymers generallypossess the following structure, PEG-W-CO₂-NHS or an equivalentactivated ester, where

W=—O₂C—(CH₂)_(b)—O— b=1-5

-   -   —O—(CH₂)_(b)CO₂—(CH₂)_(c)— b=1-5, c=2-5    -   —O—(CH₂)_(b)—CO₂—(CH₂)_(c)—O— b=1-5, c=2-5        and NHS is N-hydroxysuccinimidyl. Upon hydrolysis, the resulting        released active agent, e.g., GLP-1, will possess a short tag        resulting from hydrolysis of the ester functionality of the        polymer reagent. Illustrative releasable conjugates of this type        include: mPEG-O—(CH₂)_(b)—COOCH₂C(O)—NH-GLP-1, and        mPEG-O—(CH₂)_(b)—COO—CH(CH₃)—CH₂—C(O)—NH-GLP-1, where the number        of water-soluble polymers attached to GLP-1 can be anywhere from        1 to 4, or more preferably, from 1 to 3.

Carboxyl Coupling and Resulting Conjugates

Carboxyl groups represent another functional group that can serve as apoint of attachment to the GLP-1 moiety. Structurally, the conjugatewill comprise the following:

(GLP-1)-C(O)—X-POLY

where GLP-1-C(O)˜ corresponds to a residue of a GLP-1 moiety where thecarbonyl is a carbonyl (derived from the carboxy group) of the GLP-1moiety, X is a spacer moiety, such as a heteroatom selected from O,N(H), and S, and POLY is a water-soluble polymer such as PEG, optionallyterminating in an end-capping moiety.

The C(O)—X linkage results from the reaction between a polymericderivative bearing a terminal functional group and a carboxyl-containingGLP-1 moiety. As discussed above, the specific linkage will depend onthe type of functional group utilized. If the polymer isend-functionalized or “activated” with a hydroxyl group, the resultinglinkage will be a carboxylic acid ester and X will be O. If the polymerbackbone is functionalized with a thiol group, the resulting linkagewill be a thioester and X will be S. When certain multi-arm, branched orforked polymers are employed, the C(O)X moiety, and in particular the Xmoiety, may be relatively more complex and may include a longer linkerstructure.

Polymer reagents containing a hydrazide moiety are also suitable forconjugation at a carbonyl. To the extent that the GLP-1 moiety does notcontain a carbonyl moiety, a carbonyl moiety can be introduced byreducing any carboxylic acid functionality (e.g., the C-terminalcarboxylic acid). Specific examples of polymeric reagents comprising ahydrazide moiety, along with the corresponding conjugates, are providedin Table 2, below. In addition, any polymeric reagent comprising anactivated ester (e.g., a succinimidyl group) can be converted to containa hydrazide moiety by reacting the polymer activated ester withhydrazine (NH₂—NH₂) or tert-butyl carbazate [NH₂NHCO₂C(CH₃)₃]. In thetable, the variable (n) represents the number of repeating monomericunits and “═C-(GLP-1)” represents a residue of a GLP-1 moiety followingconjugation to the polymeric reagent were the underlined C is part ofthe GLP-1 moiety. Optionally, the hydrazone linkage can be reduced usinga suitable reducing agent. While each polymeric portion [e.g.,(OCH₂CH₂)_(n) or (CH₂CH₂O)_(n)] presented in Table 2 terminates in a“CH₃” group, other groups (such as H and benzyl) can be substitutedtherefor.

TABLE 2 Carboxyl-Specific Polymeric Reagents and the GM-GLP-1 MoietyConjugate Formed Therefrom Polymeric Reagent Corresponding Conjugate

  mPEG-Hydrazine Reagent

  Hydrazone Linkage

  mPEG-Hydrazine Reagent

  Hydrazone Linkage

  mPEG-Hydrazine Reagent

  Hydrazone Linkage

  mPEG-Hydrazine Reagent

  Hydrazone Linkage

  mPEG-Hydrazone Linkage

  Hydrazine Reagent

  mPEG-Hydrazine Reagent

  Hydrazone Linkage

  mPEG-Hydrazine Reagent

  Hydrazone Linkage

  mPEG-Hydrazine Reagent

  Hydrazone Linkage

Thiol Coupling and Resulting Conjugates

Thiol groups contained within the GLP-1 moiety can serve as effectivesites of attachment for the water-soluble polymer. The thiol groupscontained in cysteine residues of the GLP-1 moiety can be reacted withan activated PEG that is specific for reaction with thiol groups, e.g.,an N-maleimidyl polymer or other derivative, as described in, forexample, U.S. Pat. No. 5,739,208, WO 01/62827, and in Table 3 below.GLP-1 moieties for use in this embodiment of the invention include thosedescribed in WO 2004/093823.

Specific examples of the reagents themselves, along with thecorresponding conjugates, are provided in Table 3 below. In the table,the variable (n) represents the number of repeating monomeric units and“—S-(GLP-1)” represents a residue of a GLP-1 moiety followingconjugation to the water-soluble polymer, where the S represents theresidue of a GLP-1 thiol group. While each polymeric portion [e.g.,(OCH₂CH₂)_(n) or (CH₂CH₂O)_(n)] presented in Table 3 terminates in a“CH₃” group, other end-capping groups (such as H and benzyl) or reactivegroups may be used as well.

TABLE 3 Thiol-Specific Polymeric Reagents and the GLP-1 Moiety ConjugateFormed Therefrom Polymeric Reagent Corresponding Conjugate

  mPEG Maleimide Reagent

  Thioether Linkage

  mPEG Maleimide Reagent

  Thioether Linkage

  mPEG Maleimide Reagent

  Thioether Linkage

  Homobifunctional mPEG Maleimide Reagent

  Thioether Linkages

  mPEG Maleimide Reagent

  Thioether Linkage

  mPEG Maleimide Reagent

  Thioether Linkage

  mPEG Forked Maleimide Reagent

  Thioether Linkage

Branched mPEG2 Maleimide Reagent Thioether Linkage

  Branched mPEG2 Maleimide Reagent

  Thioether Linkage

Branched mPEG2 Forked Maleimide Reagent Thioether Linkages

  Branched mPEG2 Forked Maleimide Reagent

  Thioether Linkages

mPEG Vinyl Sulfone Reagent Thioether Linkage

mPEG Thiol Reagent Disulfide Linkage

Homobifunctional PEG Thiol Reagent Disulfide Linkages

mPEG Disulfide Reagent Disulfide Linkage

Homobifunctional PEG Disulfide Reagent Disulfide Linkages

With respect to conjugates formed from water-soluble polymers bearingone or more maleimide functional groups (regardless of whether themaleimide reacts with an amine or thiol group on the GLP-1 moiety), thecorresponding maleamic acid form(s) of the water-soluble polymer canalso react with the GLP-1 moiety. Under certain conditions (e.g., a pHof about 7-9 and in the presence of water), the maleimide ring will“open” to form the corresponding maleamic acid. The maleamic acid, inturn, can react with an amine or thiol group of a GLP-1 moiety.Exemplary maleamic acid-based reactions are schematically shown below.POLY represents the water-soluble polymer, and ˜S-GLP-1 represents aresidue of a GLP-1 moiety, where the S is derived from a thiol group ofthe GLP-1.

Thiol PEGylation is specific for free thiol groups on the GLP-1 moiety.Typically, a polymer maleimide is conjugated to a sulfhydryl-containingGLP-1 at pHs ranging from about 6-9 (e.g., at 6, 6.5, 7, 7.5, 8, 8.5, or9), more preferably at pHs from about 7-9, and even more preferably atpHs from about 7 to 8. Generally, a slight molar excess of polymermaleimide is employed, for example, a 1.5 to 15-fold molar excess,preferably a 2-fold to 10 fold molar excess. Reaction times generallyrange from about 15 minutes to several hours, e.g., 8 or more hours, atroom temperature. For sterically hindered sulfhydryl groups, requiredreaction times may be significantly longer. Thiol-selective conjugationis preferably conducted at pHs around 7. Temperatures for conjugationreactions are typically, although not necessarily, in the range of fromabout 0° C. to about 40° C.; conjugation is often carried out at roomtemperature or less. Conjugation reactions are often carried out in abuffer such as a phosphate or acetate buffer or similar system.

With respect to reagent concentration, an excess of the polymericreagent is typically combined with the GLP-1 moiety. The conjugationreaction is allowed to proceed until substantially no furtherconjugation occurs, which can generally be determined by monitoring theprogress of the reaction over time.

Progress of the reaction can be monitored by withdrawing aliquots fromthe reaction mixture at various time points and analyzing the reactionmixture by SDS-PAGE or MALDI-TOF mass spectrometry or any other suitableanalytical method. Once a plateau is reached with respect to the amountof conjugate formed or the amount of unconjugated polymer remaining, thereaction is assumed to be complete. Typically, the conjugation reactiontakes anywhere from minutes to several hours (e.g., from 5 minutes to 24hours or more). The resulting product mixture is preferably, but notnecessarily purified, to separate out excess reagents, unconjugatedreactants (e.g., GLP-1) undesired multi-conjugated species, and free orunreacted polymer. The resulting conjugates can then be furthercharacterized using analytical methods such as MALDI, capillaryelectrophoresis, gel electrophoresis, and/or chromatography.

An illustrative GLP-1 conjugate formed by reaction with one or moreGLP-1 thiol groups may possess the following structure:

POLY-L_(0,1)-C(O)Z—Y—S—S-(GLP-1)

where POLY is a water-soluble polymer, L is an optional linker, Z is aheteroatom selected from the group consisting of O, NH, and S, and Y isselected from the group consisting of C₂₋₁₀ alkyl, C₂₋₁₀ substitutedalkyl, aryl, and substituted aryl, and —S-GLP-1 is a residue of a GLP-1moiety, where the S represents the residue of a GLP-1 thiol group. Suchpolymeric reagents suitable for reaction with a GLP-1 moiety to resultin this type of conjugate are described in U.S. Patent ApplicationPublication No. 2005/0014903, which is incorporated herein by reference.

With respect to polymeric reagents suitable for reacting with a GLP-1thiol group, those described here and elsewhere can be obtained fromcommercial sources (e.g., Nektar Therapeutics, Huntsville Ala.). Inaddition, methods for preparing polymeric reagents are described in theliterature.

Additional Conjugates and Features Thereof

As is the case for any GLP-1 polymer conjugate of the invention, theattachment between the GLP-1 moiety and water-soluble polymer can bedirect, wherein no intervening atoms are located between the GLP-1moiety and the polymer, or indirect, wherein one or more atoms arelocated between the GLP-1 moiety and polymer. With respect to theindirect attachment, a “spacer or linker moiety” serves as a linkbetween the GLP-1 moiety and the water-soluble polymer. The one or moreatoms making up the spacer moiety can include one or more of carbonatoms, nitrogen atoms, sulfur atoms, oxygen atoms, and combinationsthereof. The spacer moiety can comprise an amide, secondary amine,carbamate, thioether, and/or disulfide group. Nonlimiting examples ofspecific spacer moieties (including “X”, X¹, X², and X³) include thoseselected from the group consisting of —O—, —S—, —S—S—, —C(O)—,—C(O)—NH—, —NH—C(O)—NH—, —O—C(O)—NH—, —C(S)—, —CH₂—, —CH₂—CH₂—,—CH₂—CH₂—CH₂—, —CH₂—CH₂—CH₂—CH₂—, —O—CH₂—, —CH₂—O—, —O—CH₂—CH₂—,—CH₂—O—CH₂—, —CH₂—CH₂—O—, —O—CH₂—CH₂—CH₂—, —CH₂—O—CH₂—CH₂—,—CH₂—CH₂—O—CH₂—, —CH₂—CH₂—CH₂—O—, —O—CH₂—CH₂—CH₂—CH₂—,—CH₂—O—CH₂—CH₂—CH₂—, —CH₂—CH₂—O—CH₂—CH₂—, —CH₂—CH₂—CH₂—O—CH₂—,—CH₂—CH₂—CH₂—CH₂—O—, —C(O)—NH—CH₂—, —C(O)—NH—CH₂—CH₂—,—CH₂—C(O)—NH—CH₂—, —CH₂—CH₂—C(O)—NH—, —C(O)—NH—CH₂—CH₂—CH₂—,—CH₂—C(O)—NH—CH₂—CH₂—, —CH₂—CH₂—C(O)—NH—CH₂—, —CH₂—CH₂—CH₂—C(O)—NH—,—C(O)—NH—CH₂—CH₂—CH₂—CH₂—, —CH₂—C(O)—NH—CH₂—CH₂—CH₂—,—CH₂—CH₂—C(O)—NH—CH₂—CH₂—, —CH₂—CH₂—CH₂—C(O)—NH—CH₂—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—, —CH₂—CH₂—CH₂—CH₂—C(O)—NH—, —C(O)—O—CH₂—,—CH₂—C(O)—O—CH₂—, —CH₂—CH₂—C(O)—O—CH₂—, —C(O)—O—CH₂—CH₂—, —NH—C(O)—CH₂—,—CH₂—NH—C(O)—CH₂—, —CH₂—CH₂—NH—C(O)—CH₂—, —NH—C(O)—CH₂—CH₂—,—CH₂—NH—C(O)—CH₂—CH₂—, —CH₂—CH₂—NH—C(O)—CH₂—CH₂—, —C(O)—NH—CH₂—,—C(O)—NH—CH₂—CH₂—, —O—C(O)—NH—CH₂—, —O—C(O)—NH—CH₂—CH₂—, —NH—CH₂—,—NH—CH₂—CH₂—, —CH₂—NH—CH₂—, —CH₂—CH₂—NH—CH₂—, —C(O)—CH₂—,—C(O)—CH₂—CH₂—, —CH₂—C(O)—CH₂—, —CH₂—CH₂—C(O)—CH₂—,—CH₂—CH₂—C(O)—CH₂—CH₂—, —CH₂—CH₂—C(O)—, CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—CH₂—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—CH₂—CH₂—,—O—C(O)—NH—[CH₂]_(h)—(OCH2CH2)_(j)—, bivalent cycloalkyl group, —O—,—S—, an amino acid, —N(R⁶)—, and combinations of two or more of any ofthe foregoing, wherein R⁶ is H or an organic radical selected from thegroup consisting of alkyl, substituted alkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, aryl and substituted aryl, (h) iszero to six, and (j) is zero to 20. Other specific spacer moieties havethe following structures: —C(O)—NH—(CH₂)₁₋₆—NH—C(O)—,—NH—C(O)—NH—(CH₂)₁₋₆—NH—C(O)—, and —O—C(O)—NH—(CH₂)₁₋₆—NH—C(O)—, whereinthe subscript values following each methylene indicate the number ofmethylenes contained in the structure, e.g., (CH₂)₁₋₆ means that thestructure can contain 1, 2, 3, 4, 5 or 6 methylenes. Additionally, anyof the above spacer moieties may further include an ethylene oxideoligomer chain comprising 1 to 20 ethylene oxide monomer units [i.e.,—(CH₂CH₂O)₁₋₂₀]. That is, the ethylene oxide oligomer chain can occurbefore or after the spacer moiety, and optionally in between any twoatoms of a spacer moiety comprised of two or more atoms. Also, theoligomer chain would not be considered part of the spacer moiety if theoligomer is adjacent to a polymer segment and merely represent anextension of the polymer segment.

As indicated above, in some instances the water-soluble polymer-(GLP-1)conjugate will include a non-linear water-soluble polymer. Such anon-linear water-soluble polymer encompasses a branched water-solublepolymer (although other non linear water-soluble polymers are alsocontemplated). Thus, in one or more embodiments of the invention, theconjugate comprises a GLP-1 moiety comprising an internal or N-terminalamine covalently attached, either directly or through a spacer moietycomprised of one or more atoms, to a branched water-soluble polymer. Asused herein, an internal amine is an amine that is not part of theN-terminal amino acid (meaning not only the N-terminal amine, but anyamine on the side chain of the N-terminal amino acid).

Although such conjugates include a branched water-soluble polymerattached (either directly or through a spacer moiety) to a GLP-1 moietyat an internal amino acid of the GLP-1 moiety, additional branchedwater-soluble polymers can also be attached to the same GLP-1 moiety atother locations as well. Thus, for example, a conjugate including abranched water-soluble polymer attached (either directly or through aspacer moiety) to a GLP-1 moiety at an internal amino acid of the GLP-1moiety, can further include an additional branched water-soluble polymercovalently attached, either directly or through a spacer moietycomprised of one or more atoms, to the N-terminal amino acid residue,such as at the N-terminal amine.

As stated above, in some instances, the branched water-soluble polymermay lack a lysine residue in which the polymeric portions are connectedto amine groups of the lysine via a “—OCH₂CONHCH₂CO—” group. In stillother instances, the branched water-soluble polymer lacks a lysineresidue (wherein the lysine residue is used to effect branching).

One preferred branched water-soluble polymer comprises the followingstructure:

wherein each (n) is independently an integer having a value of from 3 to4000, or more preferably, from about 10 to 1800.

Also forming part of the invention are multi-armed polymer conjugatescomprising a polymer scaffold having 3 or more polymer arms eachsuitable for capable of covalent attachment of a GLP-1 moiety.

Exemplary conjugates in accordance with this embodiment of the inventionwill generally comprise the following structure:

R-POLY-L_(D)-GLP-1)_(y)

wherein R is a core molecule as previously described, POLY is awater-soluble polymer, L_(D) is a degradable, e.g., hydrolyzablelinkage, and y ranges from about 3 to 15.

More particularly, such a conjugate may comprise the structure:

where m is selected from 3, 4, 5, 6, 7, and 8.

In yet a related embodiment, the GLP-1 conjugate may correspond to thestructure:

where R is a core molecule as previously described, P is a spacer, Z is—O—, —NH—, or —CH₂—, —O-GLP-1 is a hydroxyl residue of a GLP-1 moiety,and y is 3 to 15. Preferably, —NH—P—Z—C(O)— is a residue of a naturallyor non-naturally occurring amino acid.

Additional exemplary conjugates in accordance with the invention areprovided in Examples 4-26 herein.

Purification

The GLP-1 polymer conjugates described herein can be purified toobtain/isolate different conjugate species. Specifically, a productmixture can be purified to obtain an average of anywhere from one, two,or three or even more PEGs per GLP-1 moiety. In one embodiment of theinvention, preferred GLP-1 conjugates are mono-conjugates. The strategyfor purification of the final conjugate reaction mixture will dependupon a number of factors, including, for example, the molecular weightof the polymeric reagent employed, the GLP-1 moiety, and the desiredcharacteristics of the product—e.g., monomer, dimer, particularpositional isomers, etc.

If desired, conjugates having different molecular weights can beisolated using gel filtration chromatography and/or ion exchangechromatography. Gel filtration chromatography may be used to fractionatedifferent GLP-1 conjugates (e.g., 1-mer, 2-mer, 3-mer, and so forth,wherein “1-mer” indicates one polymer molecule per GLP-1, “2-mer”indicates two polymers attached to GLP-1, and so on) on the basis oftheir differing molecular weights (where the difference correspondsessentially to the average molecular weight of the water-solublepolymer. While this approach can be used to separate PEG and other GLP-1polymer conjugates having different molecular weights, this approach isgenerally ineffective for separating positional isomers having differentpolymer attachment sites within the GLP-1 moiety. For example, gelfiltration chromatography can be used to separate from each othermixtures of PEG 1-mers, 2-mers, 3-mers, and so forth, although each ofthe recovered PEG-mer compositions may contain PEGs attached todifferent reactive amino groups (e.g., lysine residues) or otherfunctional groups of the GLP-1 moiety.

Gel filtration columns suitable for carrying out this type of separationinclude Superdex™ and Sephadex™ columns available from AmershamBiosciences (Piscataway, N.J.). Selection of a particular column willdepend upon the desired fractionation range desired. Elution isgenerally carried out using a suitable buffer, such as phosphate,acetate, or the like. The collected fractions may be analyzed by anumber of different methods, for example, (i) optical density (OD) at280 nm for protein content, (ii) bovine serum albumin (BSA) proteinanalysis, (iii) iodine testing for PEG content (Sims et al. (1980) Anal.Biochem, 107:60-63), and (iv) sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS PAGE), followed by staining with barium iodide.

Separation of positional isomers is typically carried out by reversephase chromatography using a reverse phase-high performance liquidchromatography (RP-HPLC) C18 column (Amersham Biosciences or Vydac) orby ion exchange chromatography using an ion exchange column, e.g., aSepharose™ ion exchange column available from Amersham Biosciences.Either approach can be used to separate polymer-GLP-1 isomers having thesame molecular weight (positional isomers).

The resulting purified compositions are preferably substantially free ofthe non-conjugated GLP-1 moiety. In addition, the compositionspreferably are substantially free of all other non-covalently attachedwater-soluble polymers.

Compositions

Compositions of Conjugate Isomers

Also provided herein are compositions comprising any one or more of theGLP-1 polymer conjugates described herein. In certain instances, thecomposition will comprise a plurality of GLP-1 polymer conjugates. Forinstance, such a composition may comprise a mixture of GLP-1 polymerconjugates having one, two, three and/or even four water-soluble polymermolecules covalently attached to sites on the GLP-1 moiety. That is tosay, a composition of the invention may comprise a mixture of monomer,dimer, and possibly even trimer or 4-mer. Alternatively, the compositionmay possess only mono-conjugates, or only di-conjugates, etc. Amono-conjugate GLP-1 composition will typically comprise GLP-1 moietieshaving only a single polymer covalently attached thereto, e.g.,preferably releasably attached. A mono-conjugate composition maycomprise only a single positional isomer, or may comprise a mixture ofdifferent positional isomers having polymer covalently attached todifferent sites within the GLP-1 moiety. For example, a mono-conjugateGLP-1 composition may contain a mixture of mono-conjugated GLP-1 specieshaving water-soluble polymer attached to either lysine26 or lysine34.Alternately, a mono-conjugate composition may possess the water-solublepolymer attached to only lysine 26, or only lysine34, or only theN-terminus.

In yet another embodiment, a GLP-1 conjugate may possess multiple GLP-1moieties covalently attached to a single multi-armed polymer having 3 ormore polymer arms. Typically, the GLP-1 moieties are each attached atthe same GLP-1 amino acid site, e.g., the N-terminus.

With respect to the conjugates in the composition, the composition willtypically satisfy one or more of the following characteristics: at leastabout 85% of the conjugates in the composition will have from one tofour polymers attached to the GLP-1 moiety; at least about 85% of theconjugates in the composition will have from one to three polymersattached to the GLP-1 moiety; at least about 85% of the conjugates inthe composition will have from one to two polymers attached to the GLP-1moiety; or at least about 85% of the conjugates in the composition willhave one polymer attached to the GLP-1 moiety (i.e., be monoPEGylated);at least about 95% of the conjugates in the composition will have fromone to four polymers attached to the GLP-1 moiety; at least about 95% ofthe conjugates in the composition will have from one to three polymersattached to the GLP-1 moiety; at least about 95% of the conjugates inthe composition will have from one to two polymers attached to the GLP-1moiety; at least about 95% of the conjugates in the composition willhave one polymers attached to the GLP-1 moiety; at least about 99% ofthe conjugates in the composition will have from one to four polymersattached to the GLP-1 moiety; at least about 99% of the conjugates inthe composition will have from one to three polymers attached to theGLP-1 moiety; at least about 99% of the conjugates in the compositionwill have from one to two polymers attached to the GLP-1 moiety; and atleast about 99% of the conjugates in the composition will have onepolymer attached to the GLP-1 moiety (i.e., be monoPEGylated).

In one or more embodiments, the conjugate-containing composition is freeor substantially free of albumin.

In one or more embodiments of the invention, a pharmaceuticalcomposition is provided comprising a conjugate comprising a GLP-1 moietycovalently attached, e.g., releasably, to a water-soluble polymer,wherein the water-soluble polymer has a weight-average molecular weightof greater than about 2,000 Daltons; and a pharmaceutically acceptableexcipient.

Control of the desired number of polymers for covalent attachment toGLP-1 is achieved by selecting the proper polymeric reagent, the ratioof polymeric reagent to the GLP-1 moiety, temperature, pH conditions,and other aspects of the conjugation reaction. In addition, reduction orelimination of the undesired conjugates (e.g., those conjugates havingfour or more attached polymers) can be achieved through purificationmean as previously described.

For example, the water-soluble polymer-(GLP-1) moiety conjugates can bepurified to obtain/isolate different conjugated species. Specifically,the product mixture can be purified to obtain an average of anywherefrom one, two, three, or four PEGs per GLP-1 moiety, typically one, twoor three PEGs per GLP-1 moiety. In one or more embodiments, the productmixture contains one PEG per GLP-1, where PEG is releasably (viahydrolysis) attached to PEG polymer, e.g., a branched or straight chainPEG polymer.

Pharmaceutical Compositions

Optionally, a GLP-1 conjugate composition of the invention willcomprise, in addition to the GLP-1 conjugate, a pharmaceuticallyacceptable excipient. More specifically, the composition may furthercomprise excipients, solvents, stabilizers, membrane penetrationenhancers, etc., depending upon the particular mode of administrationand dosage form.

Pharmaceutical compositions of the invention encompass all types offormulations and in particular those that are suited for injection,e.g., powders or lyophilates that can be reconstituted as well asliquids, as well as for inhalation. Examples of suitable diluents forreconstituting solid compositions prior to injection includebacteriostatic water for injection, dextrose 5% in water,phosphate-buffered saline, Ringer's solution, saline, sterile water,deionized water, and combinations thereof. With respect to liquidpharmaceutical compositions, solutions and suspensions are envisioned.Compositions suitable for pulmonary administration will be described ingreater detail below.

Exemplary pharmaceutically acceptable excipients include, withoutlimitation, carbohydrates, inorganic salts, antimicrobial agents,antioxidants, surfactants, buffers, acids, bases, and combinationsthereof.

Representative carbohydrates for use in the compositions of the presentinvention include sugars, derivatized sugars such as alditols, aldonicacids, esterified sugars, and sugar polymers. Exemplary carbohydrateexcipients suitable for use in the present invention include, forexample, monosaccharides such as fructose, maltose, galactose, glucose,D-mannose, sorbose, and the like; disaccharides, such as lactose,sucrose, trehalose, cellobiose, and the like; polysaccharides, such asraffinose, melezitose, maltodextrins, dextrans, starches, and the like;and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitolsorbitol (glucitol), pyranosyl sorbitol, myoinositol and the like.Preferred, in particular for formulations intended for inhalation, arenon-reducing sugars, sugars that can form a substantially dry amorphousor glassy phase when combined with the composition of the presentinvention, and sugars possessing relatively high glass transitiontemperatures, or Tgs (e.g., Tgs greater than 40° C., or greater than 50°C., or greater than 60° C., or greater than 70° C., or having Tgs of 80°C. and above). Such excipients may be considered glass-formingexcipients.

Additional excipients include amino acids, peptides and particularlyoligomers comprising 2-9 amino acids, or 2-5 mers, and polypeptides, allof which may be homo or hetero species. Representative amino acidsinclude glycine (gly), alanine (ala), valine (val), leucine (leu),isoleucine (ile), methionine (met), proline (pro), phenylalanine (phe),tryptophan (trp), serine (ser), threonine (thr), cysteine (cys),tyrosine (tyr), asparagine (asp), glutamic acid (glu), lysine (lys),arginine (arg), histidine (his), norleucine (nor), and modified formsthereof.

Also useful as excipients, e.g., in inhalable compositions, are di- andtripeptides containing two or more leucyl residues, as described inNektar Therapeutics' International patent application WO 01/32144,incorporated herein by reference in its entirety.

Also preferred are di- and tripeptides having a glass transitiontemperature greater than about 40° C., or greater than 50° C., orgreater than 60° C., or greater than 70° C.

Although less preferred due to their limited solubility in water,additional stability and aerosol performance-enhancing peptides for usein compositions for pulmonary administration include 4-mers and 5-merscontaining any combination of amino acids as described above. The 4-meror 5-mer may comprise two or more leucine residues. The leucine residuesmay occupy any position within the peptide, while the remaining (i.e.,non-leucyl) amino acids positions are occupied by any amino acid asdescribed above, provided that the resulting 4-mer or 5-mer has asolubility in water of at least about 1 mg/ml. In some embodiments, thenon-leucyl amino acids in a 4-mer or 5-mer are hydrophilic amino acidssuch as lysine, to thereby increase the solubility of the peptide inwater.

Exemplary protein excipients include albumins such as human serumalbumin (HSA), recombinant human albumin (rHA), gelatin, casein,hemoglobin, and the like. The compositions may also include a buffer ora pH-adjusting agent, typically but not necessarily a salt prepared froman organic acid or base. Representative buffers include organic acidsalts of citric acid, ascorbic acid, gluconic acid, carbonic acid,tartaric acid, succinic acid, acetic acid, or phthalic acid. Othersuitable buffers include Tris, tromethamine hydrochloride, borate,glycerol phosphate, and phosphate. Amino acids such as glycine are alsosuitable.

The compositions of the present invention may also include one or moreadditional polymeric excipients/additives, e.g., polyvinylpyrrolidones,derivatized celluloses such as hydroxymethylcellulose,hydroxyethylcellulose, and hydroxypropylmethylcellulose, Ficolls (apolymeric sugar), hydroxyethylstarch (HES), dextrates (e.g.,cyclodextrins, such as 2-hydroxypropyl-β-cyclodextrin andsulfobutylether-β-cyclodextrin), polyethylene glycols, and pectin.

The compositions may further include flavoring agents, taste-maskingagents, inorganic salts (e.g., sodium chloride), antimicrobial agents(e.g., benzalkonium chloride), sweeteners, antioxidants, antistaticagents, surfactants (e.g., polysorbates such as “TWEEN 20” and “TWEEN80,” and pluronics such as F68 and F88, available from BASF), sorbitanesters, lipids (e.g., phospholipids such as lecithin and otherphosphatidylcholines, phosphatidylethanolamines, although preferably notin liposomal form), fatty acids and fatty esters, steroids (e.g.,cholesterol), and chelating agents (e.g., zinc and other such suitablecations). The use of certain di-substituted phosphatidylcholines forproducing perforated microstructures (i.e., hollow, porous microspheres)may also be employed.

Other pharmaceutical excipients and/or additives suitable for use in thecompositions according to the present invention are listed in“Remington: The Science & Practice of Pharmacy,” 21^(st) ed., Williams &Williams, (2005), and in the “Physician's Desk Reference,” 60th ed.,Medical Economics, Montvale, N.J. (2006).

The amount of the GLP-1 conjugate (i.e., the conjugate formed betweenthe active agent and the polymeric reagent) in the composition will varydepending on a number of factors, but will optimally be atherapeutically effective amount when the composition is stored in aunit dose container (e.g., a vial). In addition, a pharmaceuticalpreparation, if in solution form, can be housed in a syringe. Atherapeutically effective amount can be determined experimentally byrepeated administration of increasing amounts of the conjugate in orderto determine which amount produces a clinically desired endpoint.

The amount of any individual excipient in the composition will varydepending on the activity of the excipient and particular needs of thecomposition. Typically, the optimal amount of any individual excipientis determined through routine experimentation, i.e., by preparingcompositions containing varying amounts of the excipient (ranging fromlow to high), examining the stability and other parameters, and thendetermining the range at which optimal performance is attained with nosignificant adverse effects.

Generally, however, the excipient or excipients will be present in thecomposition in an amount of about 1% to about 99% by weight, from about5% to about 98% by weight, from about 15 to about 95% by weight of theexcipient, or with concentrations less than 30% by weight. In general, ahigh concentration of the GLP-1 moiety is desired in the finalpharmaceutical formulation.

Combination of Actives

A composition of the invention may also comprise a mixture ofwater-soluble polymer-(GLP-1) moiety conjugates and unconjugated GLP-1,to thereby provide a mixture of fast-acting and long-acting GLP-1.Alternatively, the composition may also comprise, in addition to a GLP-1water-soluble polymer conjugate, insulin, e.g., a basal insulin such asan acylated basal insulin or a pI-shifted basal insulin. Generally, abasal insulin is one exhibiting a prolonged time of action of greaterthan about 8 hours in a standard model of diabetes. Exemplary basalinsulins include NPH, NPL, PZI, Ultralente, and insulin glargine.

Additional pharmaceutical compositions in accordance with the inventioninclude those comprising, in addition to an extended-action GLP-1water-soluble polymer conjugate as described herein, a rapid actingGLP-1 polymer conjugate where the water-soluble polymer is releasablyattached to the side-chain of the terminal histidine (His7) at theimidazole nitrogen.

Compositions for Pulmonary Administration

Compositions comprising a GLP-1 polymer conjugate include those suitablefor pulmonary administration. The preparation and features of suchinhalable compositions will now be described.

One embodiment of the present invention provides dry powder compositionssuitable for pulmonary delivery. Dry powder compositions of the presentinvention may be prepared by any of a number of drying techniques,including by spray drying. Spray drying of the compositions is carriedout, for example, as described generally in the “Spray Drying Handbook,”5^(th) ed., K. Masters, John Wiley & Sons, Inc., NY, N.Y. (1991), and inPlatz, R., et al., International Patent Publication Nos. WO 97/41833(1997) and WO 96/32149 (1996).

A suspension or solution comprising a GLP-1 conjugate of the presentinvention can be spray-dried in a conventional spray drier, such asthose available from commercial suppliers such as Niro A/S (Denmark),Buchi (Switzerland) and the like, resulting in a dispersible, drypowder. Desirable conditions for spray drying will vary depending uponthe composition components, and are generally determined experimentally.The gas used to spray dry the material is typically air, although inertgases such as nitrogen or argon are also suitable. Moreover, thetemperature of both the inlet and outlet of the gas used to dry thesprayed material is such that it does not cause degradation of thepharmaceutical protein in the sprayed material. Such temperatures aretypically determined experimentally, although in general the inlettemperature will range from about 50° C. to about 200° C., while theoutlet temperature will range from about 30° C. to about 150° C.Parameters may include atomization pressures ranging from about 20-150psi, or from about 30-100 psi. Typically the atomization pressureemployed will be one of the following (psi): 20, 30, 40, 50, 60, 70, 80,90, 100, 110, or 120 or above.

Respirable compositions of the present invention having the featuresdescribed herein may also be produced by drying certain compositioncomponents, which result in formation of a perforated microstructurepowder as described in WO 99/16419. The perforated microstructurepowders typically comprise spray-dried, hollow microspheres having arelatively thin porous wall defining a large internal void. Theperforated microstructure powders may be dispersed in a selectedsuspension media (such as a non-aqueous and/or fluorinated blowingagent) to provide stabilized dispersions prior to drying. The use ofrelatively low density perforated (or porous) microstructures ormicroparticulates significantly reduces attractive forces between theparticles, thereby lowering the shear forces, increasing the flowabilityand dispersibility of the resulting powders, and reducing thedegradation by flocculation, sedimentation or creaming of the stabilizeddispersions thereof.

Alternatively, powders may be prepared by lyophilization, vacuum drying,spray freeze drying, super critical fluid processing (e.g., as describedin Hanna, et al., U.S. Pat. No. 6,063,138), air drying, or other formsof evaporative drying.

Dry powders may also be prepared by blending, grinding, sieving, or jetmilling composition components in dry powder form.

Once formed, the dry powder compositions are preferably maintained underdry (i.e., relatively low humidity) conditions during manufacture,processing, and storage. Irrespective of the drying process employed,the process will preferably result in inhalable, highly dispersibleparticles comprising the GLP-1 conjugates of the present invention.

In one or more embodiments, powders of the present invention may becharacterized by several features, most notably, (i) consistently highdispersibilities, which are maintained, even upon storage, (ii) smallaerodynamic particles sizes (MMADs), (iii) improved fine particle dosevalues, i.e., powders having particles sized less than 10 microns, allof which contribute to the improved ability of the powder to penetrateto the tissues of the lower respiratory tract (i.e., the alveoli) fordelivery to the systemic circulation. These physical characteristics ofthe inhalable powders of the present invention, to be described morefully below, play a role in maximizing the efficiency of aerosolizeddelivery of such powders to the deep lung.

The particles of the present invention may generally have a mass mediandiameter (MMD), or volume median geometric diameter (VMGD), or massmedian envelope diameter (MMED), or a mass median geometric diameter(MMGD), of less than about 20 μm, or less than about 10 μm, or less thanabout 7.5 μm, or less than about 4 μm, or less than about 3.3 μm, andusually are in the range of 0.1 μm to 5 μm in diameter. Preferredpowders are composed of particles having an MMD, VMGD, MMED, or MMGDfrom about 1 to 5 μm. In some cases, the powder will also containnon-respirable carrier particles such as lactose, where thenon-respirable particles are typically greater than about 40 microns insize.

The powders of the present invention may also be characterized by anaerosol particle size distribution—mass median aerodynamic diameter(MMAD)—typically having MMADs less than about 10 μm, such as less than 5μm, less than 4.0 μm, less than 3.3 μm, or less than 3 μm. The massmedian aerodynamic diameters of the powders will typically range fromabout 0.1-5.0 μm, or from about 0.2-5.0 μm MMAD, or from about 1.0-4.0μm MMAD, or from about 1.5 to 3.0 μm. Small aerodynamic diameters may beachieved by a combination of optimized spray drying conditions andchoice and concentration of excipients.

The powders of the present invention may also be characterized by theirdensities. The powder will generally possess a bulk density from about0.1 to 10 g/cubic centimeter, or from about 0.1-2 g/cubic centimeter, orfrom about 0.15-1.5 g/cubic centimeter. In one embodiment of the presentinvention, the powders have big and fluffy particles with a density ofless than about 0.4 g/cubic centimeter and an MMD between 5 and 30microns. It is worth noting that the relationship of diameter, densityand aerodynamic diameter can be determined by the following formula(Gonda, “Physico-chemical principles in aerosol delivery,” in Topics inPharmaceutical Sciences 1991, Crommelin, D. J. and K. K. Midha, Eds.,Medpharm Scientific Publishers, Stuttgart, pp. 95-117, 1992).

The powders may have a moisture content below about 20% by weight,usually below about 10% by weight, or below about 5% by weight. Such lowmoisture-containing solids tend to exhibit a greater stability uponpackaging and storage.

Additionally, the spray drying methods and stabilizers described hereinare generally effective to provide highly dispersible compositions.Generally, the emitted dose (ED) of these powders is greater than 30%,and usually greater than 40%. In some embodiments, the ED of the powdersof the present invention is greater than 50%, 60%, 70%, or higher.

A particular characteristic which usually relates to improveddispersibility and handling characteristics is the product rugosity.Rugosity is the ratio of the specific area (e.g., as measured by BET,molecular surface adsorption, or other conventional technique) and thesurface area calculated from the particle size distribution (e.g., asmeasured by centrifugal sedimentary particle size analyzer, Horiba Capa700) and particle density (e.g., as measured by pycnometry), assumingnon-porous spherical particles. Rugosity may also be measured by airpermeametry. If the particles are known to be generally nodular inshape, as is the case in spray drying, rugosity is a measure of thedegree of convolution or folding of the surface. This may be verifiedfor powders made by the present invention by SEM analysis. A rugosity of1 indicates that the particle surface is spherical and non-porous.Rugosity values greater than 1 indicate that the particle surface isnon-uniform and convoluted to at least some extent, with higher numbersindicating a higher degree of non-uniformity. The powders of the presentinvention typically have a rugosity of at least about 2, such as atleast about 3, at least about 4, or at least about 5, and may range from2 to 10, such as from 4 to 8, or from 4 to 6.

In some embodiments of the invention, powder surface area, measured bynitrogen adsorption, typically ranges from about 6 m²/g to about 13m²/g, such as from about 7 m²/g to about 10 m²/g. The particles oftenhave a convoluted “raisin” structure rather than a smooth sphericalsurface.

A particularly preferred embodiment of the present invention is onewhere at least the outermost regions, including the outer surface, ofthe powder particles are in an amorphous glassy state. It is thoughtthat when the particles have a high T_(g) material at their surfaces,the powder will be able to take up considerable amounts of moisturebefore lowering the T_(g) to the point of instability (T_(g)-T_(s) ofless than about 10° C.).

The compositions described herein typically possess good stability withrespect to both chemical stability and physical stability, i.e., aerosolperformance over time. Generally, with respect to chemical stability,the GLP-1 conjugate contained in the composition will degrade by no morethan about 10% upon spray drying. That is to say, the powder willgenerally possess at least about 90%, or about 95%, or at least about97% or greater of the intact pharmaceutical protein.

With respect to aerosol performance, compositions of the presentinvention are generally characterized by a drop in emitted dose of nomore than about 20%, or no more than about 15%, or no more than about10%, when stored under ambient conditions for a period of three months.

Alternatively, the GLP-1 polymer conjugates of the present invention canbe formulated into perforated microstructures. Such microstructures, andmethods of their manufacture, are described in U.S. Pat. No. 6,565,885.

Briefly, such perforated microstructures generally comprise a structuralmatrix that exhibits, defines, or comprises voids, pores, defects,hollows, spaces, interstitial spaces, apertures, perforations, or holes.The absolute shape (as opposed to the morphology) of the perforatedmicrostructure is generally not critical and any overall configurationthat provides the desired characteristics is contemplated as beingwithin the scope of the invention. Accordingly, some embodimentscomprise approximately microspherical shapes.

Administration

The GLP-1 conjugates of the invention can be administered by any of anumber of routes including without limitation, oral, rectal, nasal,topical (including transdermal, aerosol, buccal and sublingual),vaginal, parenteral (including subcutaneous, intramuscular, intravenousand intradermal), intrathecal, and pulmonary. Preferred forms ofadministration include parenteral and pulmonary. Suitable formulationtypes for parenteral administration include ready-for-injectionsolutions, dry powders for combination with a solvent prior to use,suspensions ready for injection, dry insoluble compositions forcombination with a vehicle prior to use, and emulsions and liquidconcentrates for dilution prior to administration, among others.

In-vivo data provided in Example 8 demonstrates the blood glucoselowering effect of an exemplary releasable GLP-1 conjugate of theinvention, as well as its ability to provide such an effect over anextended period of time, i.e., for more than 48 hours, when administeredby injection. In contrast, native GLP-1 underwent rapid clearance. See,for example, FIGS. 13 and 14.

In some preferred embodiments of the invention, the GLP-1 polymerconjugate compositions are administered pulmonarily, preferably byinhalation. In vivo data in support of this aspect of the invention areprovided in Examples 25 and 26, in which certain exemplary releasableGLP-1 conjugates were administered by intratracheal administration. Bothsets of data indicate that pulmonary administration of a releasableGLP-1 polymer conjugate is effective to result in suppression of bloodglucose levels over a period of time substantially extended over thatobserved for unconjugated GLP-1.

The dry powder compositions as described herein may be deliveredpulmonarily using any suitable dry powder inhaler (DPI), i.e., aninhaler device that utilizes the patient's inhaled breath as a vehicleto transport the dry powder drug to the lungs. Included are NektarTherapeutics' dry powder inhalation devices as described in Patton, J.S., et al., U.S. Pat. No. 5,458,135, Oct. 17, 1995; Smith, A. E., etal., U.S. Pat. No. 5,740,794, Apr. 21, 1998; and in Smith, A. E., etal., U.S. Pat. No. 5,785,049, Jul. 28, 1998, incorporated herein byreference. When administered using a device of this type, the powderedmedicament is contained in a receptacle having a puncturable lid orother access surface, preferably a blister package or cartridge, wherethe receptacle may contain a single dosage unit or multiple dosageunits. Convenient methods for filling large numbers of cavities (i.e.,unit dose packages) with metered doses of dry powder medicament aredescribed, e.g., in Parks, D. J., et al., International PatentPublication WO 97/41031, Nov. 6, 1997, incorporated herein by reference.

Other dry powder dispersion devices for pulmonary administration of drypowders include those described, for example, in Newell, R. E., et al,European Patent No. EP 129985, Sep. 7, 1988); in Hodson, P. D., et al.,European Patent No. EP472598, Jul. 3, 1996; in Cocozza, S., et al.,European Patent No. EP 467172, Apr. 6, 1994, and in Lloyd, L. J. et al.,U.S. Pat. No. 5,522,385, Jun. 4, 1996, incorporated herein by reference.Also suitable for delivering the dry powders of the present inventionare inhalation devices such as the Astra-Draco “TURBUHALER.” This typeof device is described in detail in Virtanen, R., U.S. Pat. No.4,668,218, May 26, 1987; in Wetterlin, K., et al., U.S. Pat. No.4,667,668, May 26, 1987; and in Wetterlin, K., et al., U.S. Pat. No.4,805,811, Feb. 21, 1989, all of which are incorporated herein byreference. Other suitable devices include dry powder inhalers such asRotahaler™ (Glaxo), Discus™ (Glaxo), Spiros™ inhaler (DuraPharmaceuticals), and the Spinhaler™ (Fisons). Also suitable are deviceswhich employ the use of a piston to provide air for either entrainingpowdered medicament, lifting medicament from a carrier screen by passingair through the screen, or mixing air with powder medicament in a mixingchamber with subsequent introduction of the powder to the patientthrough the mouthpiece of the device, such as described in Mulhauser,P., et al, U.S. Pat. No. 5,388,572, Sep. 30, 1997, incorporated hereinby reference.

The compositions of the present invention may also be delivered using apressurized, metered dose inhaler (MDI), e.g., the Ventolin™ metereddose inhaler, containing a solution or suspension of drug in apharmaceutically inert liquid propellant, e.g., a chlorofluorocarbon orfluorocarbon, as described in Laube, et al., U.S. Pat. No. 5,320,094,and in Rubsamen, R. M., et al, U.S. Pat. No. 5,672,581.

Alternatively, the compositions described herein may be dissolved orsuspended in a solvent, e.g., water or saline, and administered bynebulization. Nebulizers for delivering an aerosolized solution includethe AERx™ (Aradigm), the Ultravent™ (Mallinkrodt), the Pari LC Plus™ orthe Pari LC Star™ (Pari GmbH, Germany), the DeVilbiss Pulmo-Aide, andthe Acorn II™ (Marquest Medical Products).

In one or more embodiments of the invention, a method is provided, themethod comprising delivering a conjugate to a patient, the methodcomprising the step of administering to the patient a pharmaceuticalcomposition comprising a GLP-1 polymer conjugate as provided herein.Administration can be effected by any of the routes herein described.The method may be used to treat a patient suffering from a conditionthat is responsive to treatment with GLP-1 by administering atherapeutically effective amount of the pharmaceutical composition.

As previously stated, the method of delivering a GLP-1 polymer conjugateas provided herein may be used to treat a patient having a conditionthat can be remedied or prevented by administration of GLP-1. Subjectsin need of treatment with GLP-1 or a GLP-1 conjugate of the inventioninclude those with non-insulin dependent diabetes, insulin dependentdiabetes, stroke, myocardial infarction, obesity, catabolic changesafter surgery, functional dyspepsia, and irritable bowel syndrome. Alsoincluded are subjects requiring prophylactic treatment, e.g., subjectsat risk for developing non-insulin dependent diabetes. Additionalsubjects include those with impaired glucose tolerance or impairedfasting glucose.

Certain conjugates of the invention, e.g., releasable conjugates,include those effective to release the GLP-1 moiety, e.g., byhydrolysis, over a period of several hours or even days (e.g., 2-7 days,2-6 days, 3-6 days, 3-4 days) when evaluated in a suitable in-vivomodel. Releasable conjugates of the invention include those effective tolower blood glucose levels over an extended period of time, e.g., for atleast about 8 hours, for at least about 10 hours, for about 1-3 days orso, or for about 1-2.5 days.

In view of the above, the blood glucose of the patient may reach aminimum at a time ranging from about 2 hours to about 30 hours, such asabout 4 hours to about 24 hours, 6 hours to about 18 hours, or about 8hours to about 12 hours, after administration. In the case of pulmonaryadministration, the blood glucose of the patient may reach a minimum ata time ranging from about 2 hours to about 12 hours, such as about 4hours to about 10 hours or about 6 hours to about 8 hours. In the caseof subcutaneous administration, the blood glucose of the patient reachesa minimum at a time ranging from about 2 hours to about 30 hours, suchas about 4 hours to about 24 hours, 6 hours to about 18 hours, or about8 hours to about 12 hours.

The blood glucose of the patient may reach a minimum that ranges fromabout 25% to about 60%, such as about 30% to about 55%, 35% to about50%, or about 40% to about 45%, of blood glucose before administration,e.g., by subcutaneous or pulmonary administration. As an example, thepatient may have a blood glucose level of less than about 126 mg/dl,such as less than about 120 mg/dl, less than about 110 mg/dl, or lessthan 100 mg/dl, after administration, e.g., by subcutaneous or pulmonaryadministration.

The patient may have reduced blood glucose up to about 160 hours, suchas up to about 140 hours, up to about 120 hours, up to about 100 hours,up to about 80 hours, up to about 60 hours, up to about 40 hours, up toabout 24 hours, or up to about 12 hours, after administration. In thecase of pulmonary administration, the patient may have reduced bloodglucose up to about 60 hours, up to about 50 hours, up to about 40hours, up to about 30 hours, up to about 24 hours, or up to about 12hours.

The actual dose of the GLP-1 conjugate to be administered will varydepending upon the age, weight, and general condition of the subject aswell as the severity of the condition being treated, the judgment of thehealth care professional, and conjugate being administered.Therapeutically effective amounts are known to those skilled in the artand/or are described in the pertinent reference texts and literature.Generally, a conjugate of the invention will be delivered such thatplasma levels of a GLP-1 moiety are within a range of about 5picomoles/liter to about 200 picomoles/liter.

On a weight basis, a therapeutically effective dosage amount of a GLP-1conjugate as described herein will range from about 0.01 mg per day toabout 1000 mg per day for an adult. For example, dosages may range fromabout 0.1 mg per day to about 100 mg per day, or from about 1.0 mg perday to about 10 mg/day. On an activity basis, corresponding doses basedon international units of activity can be calculated by one of ordinaryskill in the art.

The unit dosage of any given conjugate (again, such as provided as partof a pharmaceutical composition) can be administered in a variety ofdosing schedules depending on the judgment of the clinician, needs ofthe patient, and so forth. The specific dosing schedule will be known bythose of ordinary skill in the art or can be determined experimentallyusing routine methods. Exemplary dosing schedules include, withoutlimitation, administration five times a day, four times a day, threetimes a day, twice daily, once daily, three times weekly, twice weekly,once weekly, twice monthly, once monthly, and any combination thereof.Once the clinical endpoint has been achieved, dosing of the compositionis halted.

It is to be understood that while the invention has been described inconjunction with the preferred specific embodiments thereof, that theforegoing description as well as the examples that follow are intendedto illustrate and not limit the scope of the invention. Other aspects,advantages and modifications within the scope of the invention will beapparent to those skilled in the art to which the invention pertains.

All articles, books, patents and other publications referenced hereinare hereby incorporated by reference in their entireties.

EXPERIMENTAL

The practice of the invention will employ, unless otherwise indicated,conventional techniques of organic synthesis and the like, which arewithin the skill of the art. Such techniques are fully explained in theliterature. Reagents and materials are commercially available unlessspecifically stated to the contrary. See, for example, J. March,Advanced Organic Chemistry Reactions Mechanisms and Structure, 4th Ed.(New York: Wiley-Interscience, 1992), supra.

In the following examples, efforts have been made to ensure accuracywith respect to numbers used (e.g., amounts, temperatures, etc.) butsome experimental error and deviation should be accounted for. Unlessindicated otherwise, temperature is in degrees C. and pressure is at ornear atmospheric pressure at sea level.

Although other abbreviations known by one having ordinary skill in theart will be referenced, other reagents and materials will be used, andother methods known by one having ordinary skill in the art will beused, the following list and methods description is provided for thesake of convenience.

Abbreviations:

mPEG-SPA mPEG-succinimidyl propionate

mPEG-SBA mPEG-succinimidyl butanoate

mPEG-OPSS mPEG-orthopyridyl-disulfide

mPEG-MAL mPEG-maleimide, CH₃O—(CH₂CH₂O)_(n)—CH₂CH₂-MAL

mPEG-SMB mPEG-succinimidyl α-methylbutanoate,CH₃O—(CH₂CH₂O)_(n)—CH₂CH₂—CH(CH₃)—C(O)—O-succinimide

mPEG-ButyrALDH₃O—(CH₂CH₂O)_(n)—CH₂CH₂—O—C(O)—NH—(CH₂CH₂O)₄—CH₂CH₂CH₂C(O)H

mPEG-PIP CH₃O—(CH₂CH₂O)_(n)—CH₂CH₂—C(O)-piperidin-4-one

mPEG-CM CH₃O—(CH₂CH₂O)_(n)—CH₂CH₂—O—CH₂—C(O)—OH)

anh. Anhydrous

CV column volume

Fmoc 9-fluorenylmethoxycarbonyl

NaCNBH₃ sodium cyanoborohydride

HCl hydrochloric acid

HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

NMR nuclear magnetic resonance

DCC 1,3-dicyclohexylcarbodiimide

DMF dimethylformamide

DMSO dimethyl sulfoxide

DI deionized

MW molecular weight

K or kDa kilodaltons

SEC Size exclusion chromatography

HPLC high performance liquid chromatography

FPLC fast protein liquid chromatography

SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis

MALDI-TOF Matrix Assisted Laser Desorption Ionization Time-of-Flight

TLC Thin Layer Chromatography

THF Tetrahydrofuran

MATERIALS: All PEG reagents referred to in the appended examples arecommercially available unless otherwise indicated. Glucagon-likePeptide-1 (GLP-1(7-36)NH₂), “GLP-1”) used in these Examples waspurchased from American Peptide Company (Sunnyvale, Calif.).

Example 1 Preparation ofN-{di(mPEG(20,000)oxymethylcarbonylamino)fluoren-9-ylmethoxycarbonyloxy}succinimidefor Reversible PEGylation of GLP-1

A. Preparation of 2,7-di(Boc-amino)fluorene

Under an argon atmosphere, 2,7-diaminofluorene (2.45 g, 12.5 mmol) wasdissolved in 1,4-dioxane (28 mL). Deionized water (14 mL), NaOH 2M (2.2eq, 27.5 mmol, 13.8 mL) and di-tert-butyldicarbonate (BOC₂O) (2.5 eq,31.3 mmol, 6.82 g) were added successively. The reaction was stirredvigorously for 20 hours at room temperature. Product precipitated as abrown solid. The reaction was quenched by the addition of water andacidification to pH 3 with KHSO₄ 1M. Product was extracted withchloroform (3×400 mL) and the combined organic layers were washed with ½saturated brine, dried over Na₂SO₄ and evaporated. Product was purifiedby flash chromatography: silica gel 60 Å eluted with 1% methanol inchloroform. The purified yellow solid (5.1 g, ˜99%) was pure by TLC(ninhydrin stain). ¹H-NMR (CDCl₃): δ (ppm) 7.7 (bs, 2H, NH urethane);7.6 (d, 2H, Ar); 7.2 (d, 2H, Ar); 6.5 (s, 2H, Ar); 3.8 (s, 2H, CH₂); 1.5(s, 18H, Boc).

B. Preparation of 9-formyl-2,7-di(Boc-amino)fluorene

Purified 2,7-di(Boc-amino)fluorene (5 g, 12.5 mmol) (prepared from stepA, above), was dissolved in ethyl formate (50 mL) and anhydrous THF (60mL) with gentle heating. (Note: ethyl formate was stored over K₂CO₃ toremove formic acid.) The solution was cooled in an ice bath and sodiumhydride 60% in mineral oil was added portion-wise (5.5 eq, 69 mmol, 2.75g). The reaction was slowly warmed to room temperature and then heatedto 50° C. after fitting with a reflux condenser. After two hours, thereaction was cooled in an ice bath and quenched by the slow addition ofdeionized water (50 mL). The aqueous layer was adjusted to pH 5 withglacial acetic acid and extracted with ethyl acetate (2×400 mL). Thecombined organic layers were dried with Na₂SO₄, filtered and evaporatedunder reduced pressure. The crude product (dark brown solid) waspurified by flash chromatography: silica gel 60 Å step-wise gradientelution 1-5% methanol in chloroform. Yield (4.8 g, ˜90%) of a yellow tobrown solid, depending on purity. ¹H-NMR (d₆-DMSO): δ (ppm) 11.0 (s,0.9H, enol); 9.3 (2 s, 1.9H, NH urethane); 7.2-8.3 (m, Ar, C¹⁰H enol);6.5 (2 s, 0.1H, NH urethane); 4.1 (m, 0.3H, CH); 1.5 (s, 18H, Boc).

C. Preparation of 9-hydroxymethyl-2,7-di(Boc-amino)fluorene

9-Formyl-2,7-di(Boc-amino)fluorene (0.47 g, 1.1 mmol) was dissolved inanhydrous methanol (MeOH) (5 mL) under an argon atmosphere. NaBH₄ (1.2eq, 1.3 mmol, 0.05 g) was added and the reaction was stirred at roomtemperature for five hours. The reaction was diluted with deionizedwater and acidified to pH 5 with glacial acetic acid. The reaction wasextracted with ethyl acetate (2×100 mL) and the organic layers werewashed with saturated NaHCO₃ (4×20 mL) and brine (3×20 mL). The organiclayers were dried over MgSO₄, filtered and evaporated. The crudeproduct, orange solid, was purified by flash chromatography: silica gel60 Å gradient elution 1-5% methanol in chloroform (alternative gradientelution with 15-20% ethyl acetate in dichloromethane). Product was ayellow solid (0.39, 83%). ¹H-NMR (CD₃OD): δ (ppm) 7.9 (s, 0.5H, NHurethane); 7.7 (s, 2H, Ar); 7.6 (d, 2H, Ar); 7.4 (d, 2H, Ar); 4.0 (m,1H, CH); 3.9 (m, 2H, CH₂); 1.6 (s, 18H, Boc).

D. Preparation of 9-hydroxymethyl-2,7-diaminofluorene dihydrochloride

9-Hydroxymethyl-2,7-di(Boc-amino)fluorene (0.39 g, 0.9 mmol) wasdissolved in 1,4-dioxane. At 0° C. concentrated HCl (2.5 mL) was addedand the reaction was stirred for two hours at 0° C. and for one hour atroom temperature. The reaction solvents were removed at reduced pressure(45° C.). The product was dissolved in methanol and evaporated (2times). The product was dissolved in methanol (8 mL) and precipitated bythe slow addition of diethyl ether and cooling (repeat). The product wasa red-orange solid (0.25 g, 91%) that showed a single spot by TLC(chloroform/methanol/acetic acid 85:15:3, ninhydrin stain). ¹H-NMR(CD₃OD): δ (ppm) 8.1 (d, 2H, Ar); 7.8 (s, 2H, Ar); 7.5 (d, 2H, Ar); 4.3(t, 1H, CH); 4.0 (d, 2H, CH₂)

E. Preparation of 9-hydroxymethyl-2,7-di(mPEG(20,000)oxymethylcarbonylamino)fluorene

mPEG-CM(20,000) (mPEG-CM having MW=19,458; 20 g, 1.03 mmol, 3.5 eq), inanhydrous toluene (80 mL) was azeotropically distilled under reducedpressure at 60° C. on a rotary evaporator. The solids were dissolved inanhydrous dichloromethane (40 mL) under an argon atmosphere followed byaddition of N-hydroxybenzotriazole (HOBt) anhydrous (3.5 eq, 1.03 mmol,139 mg) and 1,3-dicyclohexylcarbodiimide (DCC) (3.7 eq, 1.09 mmol, 224mg). In a separate flask 9-hydroxymethyl-2,7-diaminofluorenedihydrochloride (1 eq, 0.294 mmol, 88 mg) and 4-dimethylaminopyridine(2.2 eq, 0.65 mmol, 79 mg) were dissolved in anhydrous DMF (2.5 mL).After stirring the DCC reaction for several minutes (5-15 minutes), theDMF solution of 9-hydroxymethyl-2,7-diaminofluorene was quantitativelytransferred to the DCC reaction. The reaction was stirred at roomtemperature for 27 hours before solvent was evaporated at reducedpressure. The thick syrup was dissolved in dry isopropyl alcohol (400mL, slow addition) with gentle heating. The PEG product precipitated onstanding at room temperature. Additional isopropyl alcohol was (100 mL)added while stirring at 0° C. for 30 minutes. The precipitate wasfiltered and washed with cold isopropyl alcohol/diethyl ether 7:3 (80mL) and diethyl ether. The crude product (pale yellow powder,9-(mPEG(20,000)methylester)-methyl-2,7-di(mPEG(20,000)-methylamide)fluorene)was dried under hi-vacuum (yield 18.3 g).

Under an argon atmosphere, the crude product (18.3 g) was dissolved indeionized water and adjusted to pH 12±0.1 with NaOH 1M. The hydrolysisreaction mixture was stirred at room temperature for three hours. The pHwas adjusted to 3.0 with 10% phosphoric acid. (The aqueous solution wasfiltered through a bed of celite and rinsed with water.) NaCl (60 g) wasdissolved into the aqueous solution and then extracted withdichloromethane (2×150 mL). The combined organic layers were dried overMgSO₄, filtered and evaporated at reduced pressure. The crude productwas dissolved in deionized water and desalted with ion exchange resin.Ion exchange chromatography of the PEG solution was preformed on DEAEsepharose (0.9 L) eluting with water. Fractions containing PEG werecollected. The purified product (pale yellow powder) was absent ofmPEG-CM(20,000) (HPLC analysis). Yield 7.3 g, 64% (representing thetotal amount of PEG material recovered), substitution 75% or better(representing the percentage of PEG, of the amount recovered, having thedesired functionality). ¹H-NMR (CD₂Cl₂): δ (ppm) 8.9 (s, 2H, NH amide);7.9 (s, 2H, Ar); 7.7 (m, 4H, Ar); 4.1 (m, 5H, CH₂C═O, CH); 4.0 (d, 2H,CH₂); 3.6 (s, PEG backbone); 3.3 (s, 3H, —OCH3).

F.N-{di(mPEG(20,000)oxymethylcarbonylamino)fluoren-9-ylmethoxycarbonyloxy}succinimide

9-Hydroxymethyl-2,7-di(mPEG(20,000)-methylamide)fluorene (0.5 g, 0.013mmol) in anhydrous acetonitrile (10 mL) was azeotropically distilledunder reduced pressure at 50° C. on a rotary evaporator. The solid wasdissolved in anhydrous dichloromethane (2 mL, “CH₂Cl₂”) followed byaddition of triphosgene. (Care was used to trap excess phosgene gas fromreaction with base trap) (1.4 eq, 0.018 mmol, 5 mg). After severalminutes, anhydrous pyridine (2 eq, 0.026 mmol, 2 μL of pyridine indichlormethane [2 μL pyridine/50 μL dichloromethane]) was added. At oneand one-half hours most of the reaction solvent and excess phosgene (usebase trap on vent) was evaporated with gentle warming (40° C.). Thesyrup was dissolved in anhydrous dichloromethane (2 mL) followed byaddition of N-hydroxysuccinimide (5.3 eq, 0.068 mmol, 8 mg, “NHS”) andanhydrous pyridine (3.2 eq, 0.041 mmol, 83 μL of the above (2:50)solution in dichloromethane). After hour hours, the solvent wasevaporated under an argon stream. The syrup was dissolved in anhydrousisopropyl alcohol and precipitated at room temperature. The precipitatewas filtered and washed with cold isopropyl alcohol and diethyl ether.Residual solvents were evaporated under vacuum to give a very paleyellow powder. Yield 0.4 g, 80%, substitution 73% NHS carbonate by HPLC.¹H-NMR (CD₂Cl₂): δ (ppm) 8.9 (s, 2H, NH amide); 7.9 (s, 2H, Ar); 7.7 (m,4H, Ar); 4.7 (d, 2H, CH₂); 4.3 (t, 1H, CH); 4.1 (s, 4H, CH₂C═O); 2.8 (s,4H, CH₂CH₂NHS).

Using this same procedure, polymeric reagents having other molecularweights can be prepared by substituting an mPEG-CM polymeric reagenthaving a molecular weight other than 20,000 daltons.

Example 2 Preparation of N-[2,7di(4-mPEG(10,000)aminocarbonylbutyrylamino)fluoren-9-ylmethoxycarbonyloxy]succinimide(“G2PEG2Fmoc_(20k)-NHS”)

The synthesis ofmPEG(10,000)aminocarbonylbutyrylamino)fluoren-9-ylmethoxycarbonyloxy]succinimideis represented schematically in Scheme 2 below.

A. 9-Hydroxymethyl-2,7-di(4-carboxybutyrylamino)fluorene

Under an argon atmosphere, 9-hydroxymethyl-2,7-diaminofluorenedihydrochloride (preparation described in steps A through D inExample 1) was dissolved in deionized water and adjusted to pH 8 withsaturated NaHCO₃. The mixture was diluted in half with brine and theprecipitate was extracted with ethyl acetate. The ethyl acetate layerswere dried over Na₂SO₄, filtered and evaporated for9-hydroxymethyl-2,7-diaminofluorene (brown powder, 84% isolated yield).

9-Hydroxymethyl-2,7-diaminofluorene (0.38 g, 1.7 mmol) was dissolved inanhydrous tetrahydrofuran (THF) (10 mL) and glutaric anhydride (97%, 2.2eq, 3.7 mmol, 0.435 g) was added. The reaction was stirred for 4.5 hoursand absence of amine was confirmed by TLC (ninhydrin stain, 90:10:3ethyl acetate/methanol/acetic acid). The reaction mixture was dilutedwith hexanes (10 mL), filtered and washed with 1:1 THF/hexanes thenhexanes. The crude product was dissolved in a minimal amount of methanol(1 mL) and THF (10 mL) and precipitated with addition of hexanes (10mL). The mixture was cooled (4° C.), filtered and washed with 1:1THF/hexanes then hexanes. Yield was 0.59 g (77%) of yellow-orangepowder. ¹H-NMR (CD₃OD): δ (ppm) 7.9 (s, 2H, Ar); 7.7 (d, 2H, Ar); 7.5(dd, 2H, Ar); 4.0 (t, 1H, CH); 3.9 (d, 2H, CH2); 2.5 (t, 4H, CH2); 2.4(t, 4H, CH2); 2.0 (m, 4H, CH2).

B. 9 Hydroxymethyl-2,7-di(4mPEG(10,000)-aminocarbonylbutyrylamino)fluorene

mPEG-NH2(10,000) (Mn=10,200; chromatographically purified, 12.75 g, 1.25mmol) in anhydrous toluene (100 mL) was azeotropically distilled underreduced pressure at 50° C. on a rotary evaporator. The solids weredissolved in anhydrous dichloromethane (50 mL) under an argonatmosphere. A solution of 9-hydroxymethyl-2,7-di(amidoglutaricacid)fluorene (1 eq., 0.5 mmol, 0.225 g) and N-hydroxybenzotriazole(HOBt) anhydrous (2.2 eq, 1.1 mmol, 149 mg) in anhydrous DMF (5 mL) wasquantitatively added to the PEG solution (2.5 mL DMF to rinse).1,3-Dicyclohexylcarbodiimide (DCC) (2.4 eq, 1.2 mmol, 248 mg) was thenadded to the reaction solution. The reaction was stirred at roomtemperature for 24 hours before solvent was evaporated at reducedpressure. The thick syrup was dissolved in dry isopropyl alcohol (500mL, slow addition) with gentle heating. The PEG product precipitated onstanding at room temperature. The precipitate was cooled to 10° C. forten minutes, filtered and washed with cold isopropyl alcohol (200 mL)and then diethyl ether (200 mL). The crude product (off-white powder)was dried under hi-vacuum and then dissolved in deionized water. Ionexchange chromatography of the PEG solution was preformed on POROS media(0.1 L, Boehringer-Mannheim, GmbH, Mannheim Germany) eluting with water.Fractions containing neutral PEG were collected. The purified productcontained no mPEG-NH2(10,000) (HPLC analysis). Yield 5.5 g, 53%,substitution 85% or better. 1H-NMR (CD2Cl2): δ (ppm) 8.6 (s, 2H, ArNHamide); 7.9 (s, 2H, Ar); 7.6 (m, 4H, Ar); 6.4 (bs, 2H, NH amide); 4.1(m, 1H, CH); 4.0 (d, 2H, CH2); 3.6 (s, PEG backbone); 3.3 (s, 3H,—OCH3); 2.4 (t, 4H, CH2); 2.3 (t, 4H, CH2); 2.0 (m, 4H, CH2).

C. N-[2,7 di(4mPEG(10,000)aminocarbonylbutyrylamino)fluoren-9-ylmethoxycarbonyloxy]succinimide

9-Hydroxymethyl-2,7-di(4 mPEG(10,000)-aminocarbonylbutyrylamino)fluorene(5.3 g, 0.25 mmol) in anhydrous acetonitrile (100 mL) was azeotropicallydistilled under reduced pressure at 50° C. on a rotary evaporator. Thesolid was dissolved in anhydrous dichloromethane (27 mL) followed byaddition of triphosgene (1.4 eq, 0.36 mmol, 106 mg). (Care was used totrap excess phosgene gas from reaction with base trap.). After severalminutes, anhydrous pyridine (2 eq, 0.51 mmol, 41 μL) was added. Afterone and one-half hours, most of the reaction solvent and excess phosgene(use base trap on vent) was evaporated with gentle warming (40° C.). Thesyrup was dissolved in anhydrous dichloromethane (15 mL) followed byaddition of N-hydroxysuccinimide (5.3 eq, 1.35 mmol, 155 mg, “NHS”).After 15 minutes anhydrous pyridine (3.2 eq, 0.81 mmol, 66 μL) wasadded. The reaction was stirred for two hours and the solvent wasevaporated under reduced pressure. The syrup was dissolved in anhydrousisopropyl alcohol (200 mL) and precipitated at room temperature. Theprecipitate was filtered and washed with cold isopropyl alcohol anddiethyl ether (150 mL containing 10 mg BHT). Residual solvents wereevaporated under vacuum to provide an off-white powder. Yield 5.1 g,95%, substitution ˜70% NHS carbonate by HPLC.

Another polymeric reagent was prepared using this same approach exceptmPEG-NH₂ (chromatographically purified) having a weight averagemolecular weight of about 20,000 was substituted for mPEG-NH₂(10,000).The resulting polymeric reagent had a total molecular weight of about40,000 Daltons. The name of polymeric reagent so prepared is9-hydroxymethyl-2,7-di(4 mPEG(20,000)-aminocarbonylbutyrylamino)fluorene(or “G2PEG2Fmoc_(40k)-NHS”).

Another polymeric reagent was prepared using this same approach exceptmPEG-NH₂ (prepared in high purity using conventional methods) having aweight average molecular weight of about 30,000 was substituted formPEG-NH₂(10,000). The resulting polymeric reagent had a total molecularweight of about 60,000 Daltons. The name of polymeric reagent soprepared is 9-hydroxymethyl-2,7-di(4mPEG(30,000)-aminocarbonylbutyrylamino)fluorene (or“G2PEG2Fmoc_(60k)-NHS”).

Example 3 Preparation of an Exemplary GLP-1-Polymer Conjugate Having aReleasable PEG Moiety Attached to GLP-1 Preparation ofG2PEG2Fmoc_(20k)-N^(ter)-GLP-1

An illustrative polymeric reagent, G2PEG2Fmoc20k-NHS, was covalentlyattached to the N-terminus of GLP-1, to provide a prodrug form of theprotein wherein the PEG-moiety is releasably attached. The two-armnature of the polymeric reagent provides increased stability to theGLP-1 moiety subsequent to administration, to thereby provide asustained release formulation whereby GLP-1 is released from theconjugate via hydrolysis to provide the native or unmodified GLP-1precursor. The structure of G2PEG2Fmoc_(20k)-N^(ter)-GLP-1 is providedbelow (in the structure, “GLP-1” represents a residue of GLP-1).

The polymeric reagent, G2PEG2Fmoc_(20K)-NHS, was prepared as describedabove in Example 2.

A solution of 50 mg GLP-1 (nominally 1.2276×10⁻⁵ mol) (actual purity ofGLP-1 was 98.5% (by HPLC), and the peptide content was 82.2%) in 25 mLof 20 mM sodium acetate buffer at pH 5.50 was prepared, followed byaddition of 876.8 mg of G2PEG2Fmoc_(20k)-NHS (3.0692×10⁻⁵ mol) withstirring. The solution was allowed to stir for 16 hours at roomtemperature, thereby allowing for the formation ofG2PEG2Fmoc_(20k)-N^(ter)-GLP-1, a PEGylated GLP-1 conjugate. Thereaction mixture was then acidified to pH 4.30 by 20 mM HAc. Thereaction was monitored by SDS-PAGE analysis (FIG. 1).

The G2PEG2Fmoc_(20k)-N^(ter)-GLP-1 was purified to obtain themonoPEGylated conjugate of GLP-1 by cation exchange chromatography on anÄKTA Basic System (FIG. 2) using a mobile phase of 20 mM sodium acetatebuffer at pH 4.30 (Solution A) and 20 mM sodium acetate buffer with 1 MNaCl at pH 4.30 (Solution B). The column was a Vantage L LaboratoryColumn VL (Millipore) packed with SP Sepharose High Performance ionexchange media available from Amersham Biosciences. The flow rate was 14mL/min. The solution to be purified was first loaded onto the column.The loaded product was then eluted by the mobile phase using a gradient.The following gradient was used: for retention volumes 0 mL to 550 mL,0% of the mobile phase contained solution B; for retention volumes 550mL to 1041 mL, 0% of the mobile phase contained solution B; forretention volumes 1041 mL to 1093 mL, 10% of the mobile phase containedsolution B; for retention volumes 1093 mL to 1338 mL, 100% of the mobilephase contained solution B; for retention volumes 1338 mL to 1486 mL,100% of the mobile phase contained solution B; for retention volumes1486 mL and higher, 0% of the mobile phase contained solution B. The UVabsorbance of the eluent was monitored at 215 nm. The fractioncorresponding to the G2PEG2Fmoc_(20k)-N^(ter)-GLP-1 (monoPEGylated form)peak at a retention volume of 689.3 mL was collected (FIG. 2) andlyophilized. The lyophilized powder was dissolved in 25 mL 20 mM sodiumacetate buffer at pH 4.3, and the purification process was repeatedagain under the same cation exchange chromatographic conditions. Yield:179.4 mg.

The purified G2PEG2Fmoc_(20k)-N^(ter)-GLP-1 was analyzed by SDS-PAGE(FIG. 3, Lane 2) and reverse phase HPLC (FIG. 4A). The cleavable natureof the G2PEG2Fmoc_(20k)-N^(ter)-GLP-1 conjugate in aqueous media [50 mMtris(hydroxymethyl)aminomethane (Tris) solution, pH 10, overnight at 50°C.] was also studied by both SDS-PAGE analysis (FIG. 3, Lane 3) andreverse phase HPLC (FIG. 4B), from which the complete release of GLP-1from the conjugate was observed. The column was a 100 mm×2.1 mm IDBetasil C18 column with 5 μm particles, available from Thermo ElectronCorp. Reverse phase HPLC used a mobile phase of 0.1% TFA in deionizedwater (solution C) and 0.1% TFA in acetonitrile (solution D) conductedat 37° C. The gradient used for reverse phase HPLC was as follows: fortime 0.00 to 20.00 minutes, 35% of the mobile phase contained solutionD; for time 20.00 to 21.00 minutes, 55% of the mobile phase containedsolution D; for time 21.00 to 23.00 minutes, 80% of the mobile phasecontained solution D; for time 23.00 to 24.00 minutes, 80% of the mobilephase contained solution D; for time 24.00 to 25.00 minutes, 35% of themobile phase contained solution D; for time 25.00 and above, 35% ofmobile phase contained solution D.

The N-terminal PEGylation site (His⁷) of theG2PEG2Fmoc_(20k)-N^(ter)-GLP-1 conjugate (a monoPEGylated species) wasconfirmed by MALDI-TOF analysis following protease digestion of theconjugate using Endoproteinase Glu-C from Straphylococcus aureus V8.

Example 4 Preparation of an Exemplary GLP-1-Polymer Conjugate Having aReleasable PEG Moiety Attached to GLP-1 Preparation ofG2PEG2Fmoc_(40k)-N^(ter)-GLP-1

The polymeric reagent, G2PEG2Fmoc_(40k)-NHS, was prepared as describedabove in Example 2.

A solution of 50 mg GLP-1 (nominally 1.2276×10⁻⁵ mol) (actual purity ofGLP-1 was 98.5% (by HPLC), and the peptide content was 82.2%) in 25 mLof 20 mM sodium acetate buffer at pH 5.50 was prepared, followed byaddition of 1.4971 gm of G2PEG2Fmoc_(40k)-NHS (3.0692×10⁻⁵ mol) withstirring. The solution was allowed to stir for 15 hours at roomtemperature, thereby allowing for the formation ofG2PEG2Fmoc_(40k)-N^(ter)-GLP-1, a PEGylated GLP-1 conjugate. Thereaction mixture was acidified to pH 4.00 by 2 N HAc, followed bydilution to 50 mL with 20 mM sodium acetate buffer at pH 4.00.

The G2PEG2Fmoc_(40k)-N^(ter)-GLP-1 was purified to obtain themonoPEGylated conjugate of GLP-1 by cation exchange chromatography on anÄKTA Basic System (FIG. 5). The column was a Vantage L Laboratory ColumnVL (Millipore) packed with SP Sepharose High Performance ion exchangemedia (Amersham Biosciences). The flow rate in the column was 14 mL/min.The mobile phase used for the purification consisted 20 mM sodiumacetate buffer at pH 4.00 (solution A) and 20 mM sodium acetate bufferwith 1 M NaCl at pH 4.00 (solution B). The solution to be purified wasfirst loaded onto the column. The loaded product was then eluted by themobile phase using a gradient. The following gradient was used: forretention volumes 0 mL to 550 mL, 0% of the mobile phase containedsolution B; for retention volumes 550 mL to 1041 mL, 0% of the mobilephase contained solution B; for retention volumes 1041 mL to 1093 mL,10% of the mobile phase contained solution B; for retention volumes 1093mL to 1338 mL, 100% of the mobile phase contained solution B; forretention volumes 1338 mL to 1486 mL, 100% of the mobile phase containedsolution B; for retention volumes 1486 mL and higher, 0% of the mobilephase contained solution B. The UV absorbance of the eluent wasmonitored at 215 nm. The fraction corresponding to monoG2PEG2Fmoc_(40k)-N^(ter)-GLP-1 peak at retention volume of 668.4 mL wascollected (FIG. 5) and lyophilized. The lyophilized powder was dissolvedin 25 mL 20 mM sodium acetate buffer at pH 4.0, and the purificationprocess was repeated again under the same cation exchangechromatographic conditions. The collection fraction at 668 mL waslyophilized.

The purified G2PEG2Fmoc_(40k)-N^(ter)-GLP-1 was analyzed by SDS-PAGE(FIG. 6, Lane 2). The cleavable nature of theG2PEG2Fmoc_(40k)-N^(ter)-GLP-1 conjugate in aqueous media The cleavablenature of the G2PEG2Fmoc_(20k)-N^(ter)-GLP-1 conjugate in aqueous media[50 mM tris(hydroxymethyl)aminomethane (Tris) solution, pH 10, overnightat 50° C.] was also studied by SDS-PAGE analysis (FIG. 6, Lane 3), fromwhich the complete release of GLP-1 from the conjugate was observed.

Example 5 Preparation of an Exemplary GLP-1-Polymer Conjugate Having aReleasable PEG Moiety Attached to GLP-1 Preparation ofG2PEG2Fmoc_(20k)-Lys-GLP-1

The exemplary releasable polymeric reagent, G2PEG2Fmoc_(20k)-NHS, wascovalently and releasably attached to a lysine position of GLP-1,referred to herein as “internal” PEGylation of GLP-1.

A solution of 30 mg GLP-1 (nominally 7.3658×10⁻⁶ mol) (actual purity ofGLP-1 was 98.5% (by HPLC), and the peptide content was 82.2%) in 24.5 mLof 20 mM sodium carbonate-bicarbonate buffer at pH 10.0 was prepared,followed by addition of 276.3 mg of G2PEG2Fmoc_(20k)-NHS (1.1049×10⁻⁵mol, prepared as described above in Example 2) with stirring. Thesolution was allowed to stir for ten minutes at room temperature. Thereaction mixture was then acidified to pH 4.30 by 2 N HAc.

To obtain the G2PEG2Fmoc_(20k)-Lys-GLP-1 in mono-PEGylated form, thereaction mixture was divided into five aliquots, and each aliquot wasindividually purified by cation exchange chromatography on an ÄKTA BasicSystem. The column was a 5 mL resin-packed HiTrap™ SP HP, available fromAmersham Biosciences, and the flow rate in the column was 5 mL/min. Themobile phase used for the purification was 20 mM sodium acetate bufferat pH 4.30 (solution A) and 20 mM sodium acetate buffer with 1 M NaCl atpH 4.30 (solution B). The mobile phase was run using a gradient. Thefollowing gradient was used: 0 mL to 118.6 mL, 0% of the mobile phasecontained solution B; for retention volumes 118.6 mL to 219.1 mL, 0% ofthe mobile phase contained solution B; for retention volumes 219.1 mL to229.2 mL, 10% of the mobile phase contained solution B; for retentionvolumes 229.2 mL to 269.4 mL, 100% of the mobile phase containedsolution B; for retention volumes 269.4 mL to 279.4 mL, 100% of themobile phase contained solution B; for retention volumes 279.4 mL andhigher, 0% of the mobile phase contained solution B. The UV absorbanceof the eluent was monitored at 215 nm. The monoPEGylated GLP-1 fractioncorresponding to the G2PEG2-Fmoc_(20k)-Lys-GLP-1 peak at a retentionvolume of 150.4 mL was collected (FIG. 7) during each purification run.The purified G2PEG2Fmoc_(20k)-Lys-GLP-1 (in the monoPEGylated GLP-1form) from each purification run was then analyzed by SDS-PAGE (FIG. 8).The collected fractions were combined and lyophilized. Yield: 41 mg.

Example 6 Preparation of an Exemplary GLP-1-Polymer Conjugate Having aReleasable PEG Moiety Attached to GLP-1 Preparation ofG2PEG2Fmoc_(40k)-Lys-GLP-1

The exemplary releasable polymeric reagent, G2PEG2Fmoc_(40k)-NHS, wascovalently and releasably attached to a lysine position of GLP-1,referred to herein as “internal” PEGylation of GLP-1.

A solution of 50 mg GLP-1 (nominally 1.2276×10⁻⁵ mol) (actual purity ofGLP-1 was 98.5% (by HPLC), and the peptide content was 82.2%) in 45 mLof 20 mM sodium carbonate-bicarbonate buffer at pH 10.0 was prepared,followed by addition of 898.0 mg of G2PEG2Fmoc_(40k)-NHS (1.8414×10⁻⁵mol, prepared as described in Example 2) with stirring. The solution wasallowed to stir for ten minutes at room temperature. The reactionmixture was then acidified to pH 4.00 by 2 N HAc.

To obtain the G2PEG2Fmoc_(40k)-Lys-GLP-1 in monoPEGylated form, theacidified reaction mixture (50 mL), was divided into 10 aliquots, andeach 5 mL aliquot was purified by cation exchange chromatography on anÄKTA Basic System. The column was a 5 mL resin-packed HiTrap™ SP HP,available from Amersham Biosciences, and the flow rate in the column was5 mL/min. The mobile phase used for the purification was 20 mM sodiumacetate buffer at pH 4.00 (A) and 20 mM sodium acetate buffer with 1 MNaCl at pH 4.00 (B). The mobile phase was run using a gradient. Thefollowing gradient was used: 0 mL to 118.6 mL, 0% of the mobile phasecontained solution B; for retention volumes 118.6 mL to 219.1 mL, 0% ofthe mobile phase contained solution B; for retention volumes 219.1 mL to229.2 mL, 10% of the mobile phase contained solution B; for retentionvolumes 229.2 mL to 269.4 mL, 100% of the mobile phase containedsolution B; for retention volumes 269.4 mL to 279.4 mL, 100% of themobile phase contained solution B; for retention volumes 279.4 mL andhigher, 0% of the mobile phase contained solution B. The UV absorbanceof the eluent was monitored at 215 nm. The monoPEGylated GLP-1 fractioncorresponding to the G2PEG2-Fmoc_(40k)-Lys-GLP-1 peak at a retentionvolume of 158.3 mL was collected (FIG. 9) during each purification run.The purified G2PEG2Fmoc_(40k)-Lys-GLP-1 (in the mono-PEGylated GLP-1form) from each purification run was analyzed by SDS-PAGE (FIG. 10). Thecollected fractions were combined, concentrated by ultrafiltration andlyophilized. Yield: 187.5 mg.

Example 7 Preparation of an Exemplary GLP-1-Polymer Conjugate Having PEGStably Attached to GLP-1 Preparation ofmPEG_(2k)-O—CH₂CH₂C(O)—HN-Lys-GLP-1

Unlike the monoPEGylated GLP-1 conjugates described in Examples 3 to 6,which are cleavable under physiological conditions to release GLP-1, themonoPEGylated mPEG_(2k)-Lys-GLP-1 described in this example is anmPEG-GLP-1 conjugate formed by a stable amide stable linkage on oneGLP-1's lysine residues. A preparation method is described below.

A solution of 100 mg GLP-1 (nominally 2.4553×10⁻⁵ mol) (actual purity ofGLP-1 was 98.5% (by HPLC), and the peptide content was 82.2%) in 90 mLof 20 mM sodium carbonate bicarbonate buffer at pH 10.0 was prepared,followed by addition of 90.7 mg of mPEG-SPA 2K (3.6829×10⁻⁵ mol) withstirring. The solution was allowed to stir for ten minutes at roomtemperature. The reaction mixture was then acidified to pH 4.30 by 2 NHAc to a final volume of 100 mL. The solution was diluted to 200 mL byaddition of deionized water, and the diluted solution had a pH of 4.30.

This monoPEGylated form of GLP-1 so formed, designated“mPEG_(2k)-Lys-GLP-1,” was purified by cation exchange chromatography onan ÄKTA Basic System. The column was a Vantage L Laboratory Column VL(Millipore) packed with SP Sepharose High Performance ion exchange media(Amersham Biosciences). The flow rate was 14 mL/min. The mobile phaseused for the purification was 20 mM sodium acetate buffer at pH 4.30(solution A) and 20 mM sodium acetate buffer with 1 M NaCl at pH 4.30(solution B). The solution to be purified was first loaded onto thecolumn. The loaded product was then eluted using a gradient mobilephase. The following gradient was used: for retention volumes 0 mL to550 mL, 0% of the mobile phase contained solution B; for retentionvolumes 550 mL to 1041 mL, 0% of the mobile phase contained solution B;for retention volumes 1041 mL to 1093 mL, 10% of the mobile phasecontained solution B; for retention volumes 1093 mL to 1338 mL, 100% ofthe mobile phase contained solution B; for retention volumes 1338 mL to1486 mL, 100% of the mobile phase contained solution B; for retentionvolumes 1486 mL and higher, 0% of the mobile phase contained solution B.The UV absorbance of the eluent was monitored at 215 nm. The fractioncorresponding to the monoPEGylated mPEG_(2k)-Lys-GLP-1 peak at aretention volume of 1098.2 mL was collected (FIG. 11), buffer exchangedinto deionized water, and lyophilized. Yield: 23 mg.

The purified monoPEGylated mPEG_(2k)-Lys-GLP-1 was analyzed by SDS-PAGE(FIG. 12, Lane 1). Its molecular weight was determined by MALDI-TOF as5499 Da.

The PEGylation site of the monoPEGylated mPEG_(2k)-Lys-GLP-1 conjugateat lysine residues (Lys²⁶ or Lys³⁴) and not at the N-terminus (His⁷) wasconfirmed by MALDI-TOF analysis following protease digestion of theconjugate using Endoproteinase LYS-C.

Example 8 In-Vivo Study in Mice to Examine the Blood-Glucose LoweringEffects of Illustrative GLP-1 Polymer Conjugates

Male diabetic mice (BKS.Cg-+Lepr db/+Lepr db/01aHsd) were purchased fromHarlan Laboratories, Ltd. (Jerusalem, Israel). The 8-9 week old animals(30-40 gm) were placed in mouse cages (two animals per cage), andallowed at least 48 hours of acclimatization before the start of thestudy.

The preparation of G2PEG2Fmoc_(20k)-N^(ter)-GLP-1 (Example 3),G2PEG2Fmoc_(40k)-N^(ter)-GLP-1 (Example 4), G2PEG2Fmoc_(20k)-Lys-GLP-1(Example 5), and G2PEG2Fmoc_(40k)-Lys-GLP-1 (Example 6), is described inthe preceding examples. Each compound was accurately weighed into aglass vial and dissolved in normal saline in order to prepare aconcentration that would accommodate for the dose (based on GLP-1equivalents) and the injection volume of 100 μL.

The study was divided into two phases: a feasibility phase and anevaluation phase.

In the feasibility phase, the feasibility of using diabetic db/db miceto test the effectiveness of GLP-1 was first evaluated. In carrying outthe feasibility phase, several groups of mice were used wherein fourmice were used in each group. Data on the baseline glucose levels weregathered for each mouse for 2-3 days prior to drug dosing. This wasperformed to identify any outliers in the group of animals. On the dayof treatment (Day 0) each animal was weighed. A time 0 day blood sample(5 to 10 μL) was collected from the tail vein. The glucose level (mg/dL)was measured using a glucose analyzer. Each animal was then dosedsubcutaneously (SC) below the skin on the back. The amount of testarticle and the dose (60 and 120 μg/mouse) administered was based on theaverage body weight of the animal, and the total volume of the dose didnot exceed 10 mL/kg. The animals were then allowed to return into theircages. Blood samples of 5 to 10 μL (<0.5% of 2 mL blood volume for a 35g mouse) were removed through a needle prick/capillary tube at thefollowing time points: −3, −2, −1, 0, 0.04, 0.16, 0.33, 1.0, 1.16 days.Each collected blood sample was tested for its glucose level. At the endof the study, the animals were humanely euthanized by carbon-dioxideasphyxiation.

In the evaluation phase, the results from the feasibility phase wereused to select the appropriate doses required to attain a sustaineddelivery of GLP-1 for a 3-5 day effect. In carrying out the evaluationphase, eight mice were used in each group. Data on the baseline glucoselevels were gathered for each mouse three days prior to drug dosing. Onthe day of treatment (Day 0) each animal was weighed. A time 0 day bloodsample (5 to 10 μL) was collected from the tail vein. The glucose level(mg/dL) was measured using a glucose analyzer. Each animal was thendosed subcutaneously (SC) below the skin on the back. The amount of testarticle administered was based on the average body weight of the animal,and the total volume of the dose did not exceed 10 mL/kg. The animalswere then allowed to return into their cages. Blood samples of 5 to 10μL (<0.5% of 2 mL blood volume for a 35 g mouse) were removed through aneedle prick/capillary tube at the following time points: −3, −2, −1, 0,0.04, 0.16, 0.33, 0.5, 1, 2, 3, 6 days. Each collected blood sample wasfor its glucose level. Food was withdrawn from the animals for the firstfour hours after dosing. At the end of the study, the animals werehumanely euthanized by carbon-dioxide asphyxiation.

Table 4 below describes the test compounds and the dose for each groupof animals.

TABLE 4 Test Compounds and Dose for Each Group of Animals Lot or Numberof Dose Reference mice per (in Treatment Nos. group μg) Negative control(saline) Baxter, lot 8 — C645028 Positive control 2 (GLP-1) American 860, 120 Peptide, lot T05128191 G2PEG2Fmoc_(20K)-Lys_((26 or 34))-GLP1 ZH071805 8 420 G2PEG2Fmoc_(40K)-Lys_((26 or 34))-GLP1 ZH 072305 8 420G2PEG2Fmoc_(20K)-N^(ter)-GLP1 ZH 082405 8 420 ZH 092105G2PEG2Fmoc_(40K)-N^(ter)-GLP1 ZH 082505 8 420 CP2F1 ZH 082505 CP2F2

The data from the study was collected and analyzed. It was noted thatthe animals tolerated the single subcutaneous dose. As illustrated inFIG. 13, the blood glucose-lowering effect of GLP-1 and each of theG2PEG2Fmoc_(20K)-Lys-GLP-1 (designated as “PEG20-Lys-GLP1” in thefigure) and G2PEG2Fmoc_(40K)-Lys-GLP-1 (designated as “PEG40-Lys-GLP1”in the figure) conjugates is confirmed. It can be seen from thepharmacodynamic (PD) measurements that GLP-1 is cleared rapidly from themouse, but that the GLP-1 conjugates release the peptide over a periodof 3 to 4 days. That is to say, the exemplary GLP-1 degradableconjugates of the invention function somewhat like a molecular pump,releasing intact GLP-1 over time by in-vivo hydrolysis. The covalentlyattached hydrophilic polymer (i.e., PEG) functions not only to stabilizethe GLP-1 in-vivo (i.e., by protecting the protein from enzymaticdegradation), but also to extend its circulating half-life by slowlyreleasing the protein into the bloodstream over an extended period of 3to 4 days. The 40 kilodalton PEG conjugate was also observed to have asmall but extended PD effect when compared to the 20 kilodalton PEGconjugate.

The data from FIG. 13 suggest that: (a) GLP-1 is released into the mouseblood from the site of injection by diffusion and by hydrolysis from thePEGylated conjugate; and (b) the blood glucose-lowering activity of thelysine conjugated PEG-GLP1 may be due to the combination of the activityof the intact conjugates and the apparent in-vivo release of the peptidefrom the subject conjugates.

FIG. 14 illustrates the blood glucose-lowering effect of GLP-1 andG2PEG2Fmoc_(20K)-N^(ter)-GLP-1 and G2PEG2Fmoc_(40K)-N^(ter)-GLP-1. It isevident from the pharmacodynamic (PD) measurements that GLP-1 is clearedrapidly from the mouse, but the PEG GLP-1 conjugates release the peptideover a period of 3 to 4 days. It is also observed that the PEG 40kilodalton conjugate had a small but extended PD effect when compared tothe PEG 20 kilodalton conjugate.

This set of data (FIG. 14) suggest that: (a) GLP-1 is released into themouse blood from the site of injection by diffusion and by hydrolysisfrom the PEGylated conjugate; and (b) the histidine conjugated PEG-GLP1is not active, and the blood glucose-lowering activity observed is theresult of release of the peptide from the conjugate.

This study demonstrates that one injection of PEGylated GLP-1 asdescribed herein can be used to control diabetes over an extended periodof more than 48 hours. This study also demonstrates the sustainedrelease property of the G2PEG2Fmoc reagents when conjugated to GLP-1.This study also showed that GLP-1 can be PEGylated at the N-terminus toprovide a product suitable for parenteral administration.

Example 9 PEGylation of GLP-1 with Branched mPEG-N-HydroxysuccinimideDerivative, 40 kDa

Branched mPEG-N-Hydroxysuccinimide Derivative, 40 kDa, (“mPEG2-NHS”)

mPEG2-NHS, 40 kDa, stored at −20° C. under argon, is warmed to ambienttemperature. A five-fold excess (relative to the amount of GLP-1 in ameasured aliquot of the stock GLP-1 solution) of the warmed mPEG2-NHS isdissolved in 2 mM HCl to form a 10% reagent solution. The 10% reagentsolution is quickly added to the aliquot of stock GLP-1 solution (1mg/mL in sodium phosphate buffer, pH 7.0) and mixed well. After theaddition of the PEG reagent, the pH of the reaction mixture isdetermined and adjusted to 7.0. To allow for coupling of the mPEG2-NHSto GLP-1 via an amide linkage, the reaction solution is placed on a SlowSpeed Lab Rotator overnight to facilitate conjugation at roomtemperature. The reaction is quenched with Tris buffer. The conjugatesolution is characterized by SEC-HPLC to determine the components of theconjugate mixture. Ion-exchange chromatography is used to purify theconjugates.

Glp-1 conjugates of varying molecular weights are similarly preparedusing mPEG2-NHS reagents of differing molecular weights: 10 kDa, 15 kDA,20 kDa, 30 kDa, 50 kDa, 60 kDa, etc.

Example 10 PEGylation of GLP-1 with Linear mPEG-Succinimidylα-Methylbutanoate Derivative, 30 kDa

Linear mPEG-Succinimidyl α-Methylbutanoate Derivative, 30 kDa(“mPEG-SMB”)

mPEG-SMB, 30 kDa, stored at −20° C. under argon, is warmed to ambienttemperature. A ten-fold excess (relative to the amount of GLP-1 in ameasured aliquot of the stock GLP-1 solution) of the warmed mPEG-SMB isdissolved in 2 mM HCl to form a 10% reagent solution. The 10% reagentsolution is quickly added to the aliquot of stock GLP-1 solution (1mg/mL in sodium phosphate buffer, pH 7.0) and mixed well. After theaddition of the mPEG-SMB, the pH of the reaction mixture is determinedand adjusted to 7.0. To allow for coupling of the mPEG-SMB to GLP-1 viaan amide linkage, the reaction solution is placed on a Slow Speed LabRotator overnight to facilitate conjugation at room temperature. Thereaction is quenched with Tris buffer.

The conjugate solution is characterized by SEC-HPLC. Ion-exchange isused to purify the conjugates.

Using this same approach, other conjugates are prepared using mPEG-SMBhaving other weight average molecular weights, e.g., 2 kD, 5 kD, 10 kD,20 kD, 40 kD, etc.

Example 11 PEGylation of GLP-1 with mPEG-Piperidone, 20 kDa

mPEG-Piperidone (mPEG-PIP) having a molecular weight of 20,000 Daltonsis obtained from Nektar Therapeutics (Huntsville, Ala.). The basicstructure of the polymeric reagent is provided below:

Linear mPEG-Piperidone Derivative, 20 kDa (“mPEG-PIP”). Bottom StructureCorresponds to Hydrated Form

mPEG-PIP, 20 kDa, stored at −20° C. under argon, is warmed to ambienttemperature. A fifty to one hundred-fold excess (relative to the amountof GLP-1 in a measured aliquot of the stock GLP-1) of the warmedmPEG-PIP is dissolved in 10 mM sodium phosphate (pH 7.0) to form a 10%reagent solution. The 10% reagent solution is quickly added to thealiquot of stock GLP-1 solution (1 mg/mL in sodium phosphate buffer, pH7.0) and mixed well. After the addition of the mPEG-PIP, the pH of thereaction mixture is determined and adjusted to 7.0, followed by mixingfor thirty minutes. A reducing agent, sodium cyanoborohydride, is thenadded to make 13 mM NaCNBH₃. The reaction solution is placed on a SlowSpeed Lab Rotator overnight to facilitate conjugation at roomtemperature. The reaction is quenched with Tris buffer.

The conjugate solution is characterized by SEC-HPLC. Ion-exchange isused to purify the conjugates.

Using this same approach, other conjugates can be prepared usingmPEG-PIP having other weight average molecular weights, e.g., 2 kD, 5kD, 10 kD, 30 kD, 40 kD, etc.

Example 12 PEGylation of GLP-1 with Linear mPEG-ButyraldehydeDerivative, 20 kDa CH₃O(CH₂CH₂O)_(n)—C(O)NH—(CH₂CH₂O)₄CH₂CH₂CH₂CHOLinear mPEG-Butyraldehyde Derivative, 20 kDa (“mPEG-ButyrALD”)

mPEG-ButyrALD, 20 kDa, stored at −20° C. under argon, is warmed toambient temperature. A thirty-fold excess (relative to the amount ofGLP-1 in a measured aliquot of the stock GLP-1) of the warmedmPEG-ButryALD is dissolved in Milli-Q H₂O to form a 10% reagentsolution. The 10% reagent solution is quickly added to the aliquot ofstock GLP-1 solution (1 mg/mL in sodium phosphate buffer, pH 7.0) andmixed well. After the addition of the mPEG-ButryALD, the pH of thereaction mixture is determined and adjusted to 6.0, followed by mixingfor thirty minutes. A reducing agent, sodium cyanoborohydride, is thenadded to make 9 mM NaCNBH₃. The reaction solution is placed on a SlowSpeed Lab Rotator overnight to facilitate conjugation at roomtemperature. The reaction is quenched with Tris buffer. The conjugatesolution is characterized by SEC-HPLC and anion-exchange chromatography.

Using this same approach, other conjugates can be prepared usingmPEG-ButyrALD having other weight average molecular weights, e.g., 2 kD,5 kD, 10 kD, 30 kD, 40 kD, etc.

Example 13 PEGylation of GLP-1 with Branched mPEG-ButyraldehydeDerivative, 40 kDa

Branched mPEG-Butyraldehyde Derivative, 40 kDa (“mPEG2-ButyrALD”)

mPEG2-ButyrALD, 40 kDa, stored at −20° C. under argon, is warmed toambient temperature. A thirty-fold excess (relative to the amount ofGLP-1 in a measured aliquot of the stock GLP-1) of the warmedmPEG2-ButryALD was dissolved in Milli-Q H₂O to form a 10% reagentsolution. The 10% reagent solution is quickly added to the aliquot ofstock GLP-1 solution (1 mg/mL in sodium phosphate buffer, pH 7.0) andmixed well. After the addition of the mPEG2-ButryALD, the pH of thereaction mixture is determined and adjusted to 6.0 using conventionaltechniques, followed by mixing for thirty minutes. A reducing agent,sodium cyanoborohydride is then added to make 9 mM NaCNBH₃. The reactionsolution is placed on a Slow Speed Lab Rotator overnight to facilitateconjugation at room temperature. The reaction is quenched with Trisbuffer.

The conjugate solution is characterized by SEC-HPLC and ion-exchangechromatography method.

Using this same approach, other conjugates can be prepared usingmPEG2-ButyrALD having other weight average molecular weights, e.g., 10kD, 15 kD, 20 kD, 30 kD, 50 kD, 60 kD, etc.

Example 14 PEGylation of GLP-1 with mPEG-SBA

mPEG-Succinimidyl butanoate having a molecular weight of 20,000 Daltonsis obtained from Nektar Therapeutics, (Huntsville, Ala.). The basicstructure of the polymer reagent is provided below:

GLP-1 is dissolved in aqueous solution (1 mg/mL phosphate bufferedsolution). To this solution is then added a 1.5 to 10-fold molar excessof mPEG-SBA. After the addition of the mPEG-SBA, the pH of the reactionmixture is determined and adjusted to 7.0 to 7.5. The resulting mixtureis stirred at room temperature for several hours.

The reaction mixture is analyzed by SDS-PAGE to determine the degree ofPEGylation of the protein.

Example 15 Conjugation of Cysteine-Inserted GLP-1 with mPEG-MAL, 20K

GLP-1 is inserted with one or more cysteine residues according asdescribed in U.S. Patent Application Ser. Nos. 60/346,474 and60/405,097. Illustrative cysteine-modified GLP-1 compounds are describedin WO 2004/093823.

mPEG-MAL, 20K, stored at −20° C. under argon, is warmed to ambienttemperature. A five- to twenty-fold excess of the warmed mPEG-MAL, 20K,is dissolved in deionized water to make a 10% mPEG MAL solution. ThemPEG MAL solution is quickly added to an aliquot of stockcysteine-modified GLP-1 solution (1 mg/mL in 50 mM HEPES, pH 7.0) and ismixed well. After one hour of reaction at room temperature, the reactionvial is transferred to the cold room and the reaction is allowed toproceed overnight at 4° C. on Rotomix (slow speed, Thermolyne).

The conjugate mixture is purified using gel filtration chromatography. Asize exclusion chromatography method is developed for analyzing thereaction mixtures, and the final products. SDS-PAGE analysis is alsoused for the characterization of the samples.

Example 16 Conjugation of GLP-1 with mPEG-MAL, 30K

Cysteine-modified GLP-1 is obtained as described in Example 16 above.

mPEG-MAL, 30K, stored at −20° C. under argon, is warmed to ambienttemperature. A five- to twenty-fold excess of the warmed mPEG-MAL, 30K,is dissolved in deionized water to make a 10% mPEG MAL solution. ThemPEG MAL solution is quickly added to an aliquot of stockcysteine-inserted GLP-1 solution (1 mg/mL in 50 mM HEPES, pH 7.0) and ismixed well. After one hour of reaction at room temperature, the reactionvial is transferred to the cold room and the reaction is allowed toproceed overnight at 4° C. on Rotomix (slow speed, Thermolyne).

The conjugate mixture is purified using gel filtration chromatography. Asize exclusion chromatography method is developed for analyzing thereaction mixtures, and the final products. SDS-PAGE analysis is alsoused for the characterization of the samples.

Example 17 PEGylation of GLP-1 with mPEG-succinimidyl Benzamid-Carbonate20 kDa (mPEG-SBC 20 kDa) in an Aqueous Reaction

mPEG-SBC 20 kDa, available from Nektar Therapeutics (Huntsville, Ala.),stored at −20° C. under argon, is warmed to ambient temperature. Thereaction is performed at room temperature. The calculated quantity ofthe warmed mPEG-SBC 20 kDa (414 mg, to obtain an 8-fold molar excess ofmPEG-SBC 20 kDa based upon absolute GLP-1 content) is weighed into a 5mL glass vial. 1.1 mL of DMSO is added to PEG, and it is heated in a 40°C. water bath until the PEG is dissolved. It is then allowed toequilibrate back to room temperature. A 2.0 mL aliquot of a 4.5 mg/mLsolution of GLP-1 prepared in phosphate buffered saline, PBS, pH 7.4) isadded to 10 mL glass vial and the volume brought to 4.5 mL withadditional PBS. The protein is stirred using a magnetic stirrer at amoderate speed. The PEG is added to the protein via a syringe infusionat a rate of approximately 1 mL/min. The reaction is allowed to proceedfor 10 minutes. It is then quenched by dropping the pH to 5.5 with 1 MHCl.

The conjugate solution is analyzed by SDS-PAGE and RP-HPLC.

Example 18 PEGylation of GLP-1 with mPEG-succinimidyl Benzamid-Carbonate30 kDa (mPEG-SBC 30 kDa) in an Aqueous Reaction

GLP-1 is PEGylated as described in Example 18 above with the exceptionthat the PEG reagent employed possesses a molecular weight of 30 kDa.

Example 19 PEGylation of GLP-1 with mPEG-succinimidyl Phenyl-Carbonate20 kDa (mPEG-SPC 20 kDa) In Aqueous Reaction

mPEG-SPC 20 kDa, available from Nektar Therapeutics (Huntsville, Ala.),stored at −20° C. under argon, is warmed to ambient temperature. Thereaction is performed at room temperature. An absolute 8-fold molarexcess of mPEG-SPC reagent is used, based upon absolute peptide content.The PEG reagent is weighed into a 5 mL glass vial containing a magneticstirrer bar. A 2.0 mL aliquot of a 4.5 mg/mL solution of GLP-1 preparedin phosphate buffered saline, PBS, pH 7.4 is added and the volumebrought to 4.5 mL with additional PBS. The mixture is stirred at maximumspeed using a magnetic stirrer until the PEG is fully dissolved. Thestirring speed is reduced to 50% and the reaction is allowed to proceedto formation of conjugate product. The pH of the conjugate solution atthe end of the reaction is measured and further acidified by addition of0.1M HCl, if necessary, to bring the pH of the final solution to about5.5.

The conjugate solution is then analyzed by SDS-PAGE and RP-HPLC (C18) todetermine the extent of reaction (i.e., whether the reaction has gone tocompletion).

Additional reactions, conducted as described above, are carried out with(i) mPEG-SPC 30 kDa, and (ii) mPEG-SPC 40 kDa, available from NektarTherapeutics, Huntsville, Ala.

Example 20 In Vitro Activity Assay of PEG-Glp-1 Conjugates

The bioactivity of the conjugates described in Examples 3-7 and 9-20 istested using an in-vitro activity assay as described in Zlokarnik, etal. (1998), Science, 279:84-88.

HEK-293 cells expressing the human GLP-1 receptor, using the PanVera LLCCRE-BLAM system, are seeded at 20,000 to 40,000 cells/well/100 μl DMEMmedium with 10% FBS into a poly-d-lysine coated 96 well plate. The dayafter seeding, the medium is flicked off and 80 μA plasma-free DMEMmedium is added. On the third day after seeding, 20 μl of plasma-freeDMEM medium with 0.5% BSA containing different concentrations ofPEG-GLP-1 conjugate is added to each well to generate a dose-responsecurve. Generally, fourteen dilutions containing from 3 nanomolar to 30nanomolar PEG-GLP-1 conjugate are used to generate a dose response curvefrom which EC50 values can be determined. After 5 hours incubation, withthe PEG-GLP-1 conjugate, 20 μl of β-lactamase substrate (CCF2/AM, PanVera LLC) is added and incubation is continued for one hour at whichtime fluorescence is determined on a fluorimeter.

Example 21 In Vitro Release Profile of G2PEG2Fmoc_(20K)-N^(ter)-GLP-1

The in vitro release profile of G2PEG2Fmoc_(20K)-N^(ter)-GLP-1 wasdetermined.

G2PEG2Fmoc_(20K)-N^(ter)-GLP-1 (in the faun of mono-PEGylated GLP-1) wasprepared as described in Example 3 and was used to evaluate the releaseof GLP-1 under hydrolysis conditions.

The conditions used to determine the in vitro release profileG2PEG2Fmoc_(20K)-N^(ter)-GLP-1 included: 2 mg/mLG2PEG2Fmoc_(20K)-N^(ter)-GLP-1 (monoPEGylated GLP-1 form) inphosphate-buffered saline, pH 7.4, 37° C. with samples taken at varioustime points and tested for the presence of “free” or unconjugated GLP-1.The release of GLP-1 was monitored by reverse phase HPLC at 215 nm.

FIG. 15 sets forth the results of the experiment is graph form, whereY=A_(t)/A_(max) (A_(t) is HPLC peak area of released GLP-1 at time of t(hr) and A_(max) is HPLC peak area of GLP-1 reached its maximumrelease). Because the reaction kinetics represent a first order reactiondue to the linearity of the plot, it can be concluded that ln1/(1−Y)=kt, where k is the slope, t_(1/2)=ln 2/k. Based uponextrapolation of the data, the conjugate was determined to possess ahydrolysis half-life of 56.8 hours.

Example 22 Synthesis and Purification of mPEG_(2k)-O(CH₂)₄-N^(ter)-GLP-1

GLP-1 (43.2 mg, 13.0 μmol) was dissolved in a sodium acetate buffersolution (20 mL, pH=5.02) and CH3CN (1000 μL). mPEG2k-butyrALD (259.2mg, 130 mop was added with stirring and the reaction was maintained atroom temperature for 20 min. Solid NaCNBH3 (˜5-8 mg, 7-10 eq) was thenadded and the reduction reaction was monitored by analytical HPLC. Afterallowing to react overnight (16 h), the product was purified by FPLC(fast protein liquid chromatography) using an ÄKTA™ Basic System(Pharmacia) and a gradient of 36.3-56% CH3CN in 0.1% TFA Milli-Q waterin 0.76 CV (column volume). Fractions corresponding to monomPEG2k-O(CH2)4-Nter-GLP-1 were collected. The product was lyophilizedand obtained as a white powder. Yield: 64.5 mg.

Example 23 Synthesis, Purification and Hydrolysis ofmPEG_(5K)-OC₆H₄—OCO-N^(ter)-GLP-1 (mPEG_(5k)-SPC-N^(ter)-GLP-1)

GLP-1 (43.6 mg, 13.2 μmol) was dissolved in a sodium acetate buffersolution (28 mL, pH=5.95) at room temperature. mPEG_(5K)-SPC (556 mg,111 μmol) was added in several different portions while the reactionprogress was monitored by analytical HPLC. After the maximum peak areaof product was reached, the product mixture was purified immediatelyusing an ÄKTA™ Basic System (Pharmacia) with a gradient of 37.8-60%CH₃CN in 0.1% TFA Milli-Q water in 5 CV. The fractions corresponding tomono mPEG_(5k)-SPC-N^(ter)-GLP-1 were collected (14 mL/fraction), andthe product was lyophilized and obtained as a white powder. Yield: 75mg.

Hydrolysis:

The mPEG_(5k)-SPC-N^(ter)-GLP-1 was dissolved in PBS buffer (pH=7.2) andwarmed to 37° C. The hydrolysis was monitored by analytical HPLC. Thehalf-life was determined to be about 8 hrs according to MS detector. ThePEGylated GLP-1 was completely hydrolyzed after 3 days, leaving 6% ofunknown product. Although native GLP-1 was released upon hydrolysis, themajor hydrolysis product was not free GLP-1 but rather a His-carbamatecompound as shown below. This result was further confirmed by LC-MS.

A similar phenomenon was observed for the counterpart N-terminal GLP-1conjugate prepared by reaction with an mPEG-SBC reagent. When attemptingto further purify the conjugate, under the conditions utilized, theconjugate was found to be extremely labile. The conjugate rapidlyhydrolyzed to native GLP-1 and a GLP-1 derivative resulting from anintramolecular ring closure reaction due to reaction at the carbamatecarbonyl by the imidazolyl nitrogen of the N-terminal histidine ofGLP-1. The modified GLP-1 thereby released is shown below. Furtherpurification was not pursued.

Example 24 Preparation of an Exemplary GLP-1-Polymer Conjugate Having aReleasable PEG Moiety Attached to GLP-1 Preparation ofPEG2-CAC-Fmoc_(4k)-N^(ter)-GLP-1

An illustrative polymeric reagent, PEG2-CAC-Fmoc_(4k)-BTC (structureprovided below), was covalently attached to the N-terminus of GLP-1 toprovide a prodrug form of the protein wherein the PEG-moiety isreleasably attached. The two-arm nature of the polymeric reagentprovides increased stability to the GLP-1 moiety subsequent toadministration, to thereby provide a sustained release formulationwhereby GLP-1 is released from the conjugate via hydrolysis to providethe native or unmodified GLP-1 precursor. The structure ofPEG2-CAC-Fmoc4k-Nter-GLP-1 is provided above (in the structure, “GLP-1”represents a residue of GLP-1 corresponding to amino acids 8-36; theN-terminal histidine of GLP-1 is shown explicitly to illustrateconjugation at the alpha amine rather than of the imidazole ring).

PEG2-CAC-Fmoc_(4k)-BTC (BTC=benzotriazole carbonate) was preparedaccording to the methods described in U.S. patent application Ser. No.11/454,971, filed on Jun. 16, 2006, to which the instant applicationclaims priority, and the contents of which have been incorporated hereinby reference.

A solution of 400 mg GLP-1 (1.1146×10⁻⁴ mol) in 100 mL of 20 mM sodiumacetate buffer at pH 5.50 was prepared, followed by slow addition over 5minutes with stirring of 1.737 g of PEG2-CAC-Fmoc_(4k)-BTC (3.9013×10⁴mol), which was freshly dissolved in 17 mL of 2 mM HCl. The solution wasallowed to stir for 13 hours at room temperature, thereby allowing forthe formation of PEG2-CAC-Fmoc_(4k)-N^(ter)-GLP-1, a PEGylated GLP-1conjugate. The reaction mixture was quenched by addition of 1 mL of 1Mglycine. The solution pH was adjusted to 7.0 by 0.5 N NaOH. The reactionmixture was allowed to stir at room temperature for another 2 hours. Thequenched reaction mixture was then acidified to pH 4.0 by addition of 20mM HAc. The reaction was monitored by reversed phase HPLC, using a 100mm×4.6 mm ID Onyx monolithic C8 column with Mesopores of 130 Å andMacropores of 2 μm, available from Phenomenex. A mobile phase of 0.1%TFA in deionized water (solution C) and 0.1% TFA in acetonitrile(solution D) at 25° C. was employed using a linear gradient from 35% Bto 55% B over 4 minutes.

To obtain the PEG2-CAC-Fmoc_(4k)-N^(ter)-GLP-1 in monoPEGylated form,the acidified reaction mixture (125 mL) was divided into 3 aliquots, andeach aliquot was individually purified by cation exchange chromatographyon an ÄKTA Basic System using a mobile phase of 20 mM sodium acetatebuffer at pH 4.30 (Solution A) and 20 mM sodium acetate buffer with 1 MNaCl at pH 4.30 (Solution B). The column utilized was a Vantage LLaboratory Column VL (Millipore) packed with 160 mL column volume of SPSepharose High Performance column media (Amersham) with a flow rate of30 mL/min. The crude solution was first diluted by addition of sodiumacetate buffer at pH 4.30, and was then loaded onto the column. The UVabsorbance of the eluent was monitored at 215 nm. The fractioncorresponding to the PEG2-CAC-Fmoc_(4k)-N^(ter)-GLP-1 (monoPEGylatedform) peak was collected. Purification of the remaining crude productwas conducted, and the fractions corresponding to thePEG2-CAC-Fmoc_(4k)-N^(ter)-GLP-1 (monoPEGylated form) peak werecombined, analyzed by reversed phase HPLC, and concentrated byultrafiltration. The concentrated solution was buffer exchanged into 20mM sodium citrate buffer at pH 4.3. The solution was then lyophilized.Yield: 163.2 mg.

The purified PEG2-CAC-Fmoc_(4k)-N^(ter)-GLP-1 was analyzed by SDS-PAGE.Its molecular weight was determined by MALDI-TOF as 7831.6 Da. Thecleavable nature of the G2PEG2Fmoc_(20k)-N^(ter)-GLP-1 conjugate inaqueous media [1:2 v/v mixture of 20 mM sodium citrate buffer at pH 4.30with 200 mM tris(hydroxymethyl)aminomethane (Tris) solution, incubated16 hours at 37° C.] was also investigated by reversed phase HPLC, fromwhich the complete release of GLP-1 from the conjugate was observed.

Example 25 Pharmacodynamic Evaluation of a Modified GLP-1 Agonist,PEG2-CAC-Fmoc_(4k)-N^(ter)-GLP-1, in a Diabetic Mouse Model

The objective of the study was to demonstrate the pharmacologicalactivity of an illustrative GLP-1 conjugate,PEG2-CAC-Fmoc_(4k)-N^(ter)-GLP-1, administered intratracheally (IT) inthe mouse.

A male diabetic mouse (BKS.Cg-+Lepr db/+Lepr db/01aHsd) model was usedin the study. The test system included 21 male diabetic mice(BKS.Cg-+Lepr db/+Lepr db/01aHsd) approximately 8-9 weeks of age (HarlanLabs). See Table 5. The mice were anesthetized and then suspendedvertically by their upper incisors. Approaching the suspended animalfrom the dorsal position, a 1 mL syringe fitted with a gavage needle wasinserted orally into the mouse and descended into the trachea just abovethe carina. The dose was instilled into the lungs and the gavage needlewas then immediately removed. Exenatide was administered by subcutaneous(SC) injection using a 20 G1 inch 1 mL tuberculin syringe. Blood samplesof approximately 0.07 mL were collected from the tail vein at each timepoint. These samples were tested for concentrations of glucose. Theglucose measurement was made in duplicate with Glucometer Elite (BayerCorp., Elkart, Ind.) glucose monitors. The upper limit of quantitation(ULOQ) of these glucometers is 600 mg/dl. This value (600 mg/dl) wasused for calculation of descriptive statistics when the ULOQ wasexceeded. Group mean blood glucose concentrations, expressed as % ofbaseline, are given in FIG. 16. These values were calculated for eachindividual animal using its 24-hour pre-dose blood glucose measurementas baseline. Statistical evaluation was made with a 2-tailed t-test at aP value of 0.05 and compared group means of treatment and vehicle groupsat corresponding time points. In the absence of a corresponding vehicletime point for the 1 hour exenatide measurement, the 4 hour vehiclemeasurement was used for statistical comparison. Selected time points ofPEG2-CAC-Fmoc_(4k)-N^(ter)-GLP-1 and exenatide-treated groups were alsocompared to their respective T=0 values.

TABLE 5 Study Design Total Dose of Test Route of Number of Article No.of Group Adminis- Animals/ (μg/ Dosing No. Control/Test Article trationGender mouse) Days 1 PEG2-CAC- IT 12 M  1500 1 Fmoc_(4k)- N^(ter)-GLP-12 Vehicle IT 6 M NA 1 3 Exenatide SC 3 M 0.1 1

The objective of this study, i.e., to demonstrate pharmacologicalactivity of an illustrative releasable GLP-1 conjugate, whenadministered intratracheally (IT) in the mouse, was achieved.

Mice were administered PEG2-CAC-Fmoc_(4k)-N^(ter)-GLP-1 or vehicleintratracheally and were tested for blood glucose concentrations atvarious time points post-administration. Exenatide administered SC wasused as a positive control.

A significant reduction in blood glucose was observed at the 1 hour timepoint in the group administered exenatide. Blood glucose had returned tobaseline by the next time point (8 hrs).

PEG2-CAC-Fmoc_(4k)-N^(ter)-GLP-1 caused a substantial reduction in bloodglucose at 4 and 8 hours after administration. The magnitude of theglucose suppression was greater than that observed followingadministration of exenatide. This reduction was statisticallysignificant both when compared to the T=0 hour pre-dose measurement andto the values for the vehicle group at the corresponding time point.

Intact PEG2-CAC-Fmoc_(4k)-N^(ter)-GLP-1 is thought not to havepharmacological activity. This is presumably due to the interference ofthe N-terminal PEG with the interaction of the GLP-1 ligand to itsreceptor. The observation of in vivo pharmacological activity and theoccurrence of the activity maximum 8 hours after administration areconsistent with in vivo cleavage of PEG2-CAC-Fmoc_(4k)-N^(ter)-GLP-1 andrelease of active GLP-1.

At 3 other time points (31, 52, and 55 hours), a statisticallysignificant difference was observed between thePEG2-CAC-Fmoc_(4k)-N^(ter)-GLP-1 and vehicle groups. The magnitude ofthe difference was considerably smaller than that observed at 4 and 8hours. In contrast to the reductions observed at 4 and 8 hours, theglucose values at these 3 time points were not significantly differentfrom the values at T=0. The biological significance of this observationwill need to be further evaluated but a delayed cleavage of residual4K-FMOC GLP-1 in the lung could theoretically result in delayed glucosesuppression.

No adverse effect attributed to compound was observed in these studies.The one mouse death (animal 1-8) was attributed to the administrationprocess.

In sum, the results of this experiment indicate that the intratrachealadministration of PEG2-CAC-Fmoc_(4k)-N^(ter)-GLP-1 resulted in astatistically significant, profound and prolonged suppression of bloodglucose in a male diabetic mouse (BKS.Cg-+Lepr db/+Lepr db/01aHsd)model.

Example 26 Pharmacodynamic Evaluation of a Modified GLP-1 Agonist,mPEGSPC_(5k)-N^(ter)-GLP-1, in a Diabetic Mouse Model

The objective of this study was to demonstrate the pharmacologicalactivity of the exemplary, releasable GLP-1 conjugate,mPEGSPC_(5k)-N^(ter)-GLP-1, when administered intratracheally (IT) inthe mouse.

A male diabetic mouse (BKS.Cg-+Lepr db/+Lepr db/01aHsd) model was usedin the study. The conjugate administered, mPEGSPC_(5k)-N^(ter)-GLP-1, iscleavable and was expected to release active GLP-1 in vivo. The releasedGLP-1 moiety was expected to have an approximately 200-fold reducedactivity due to a residual carbonyl residue at the N-terminus (See,e.g., Example 23 above).

The test system included 18 male diabetic mice (BKS.Cg-+Lepr db/+Leprdb/01 aHsd) approximately 8-9 weeks of age (Harlan Labs). The mice wereanesthetized and then suspended vertically by their upper incisors.Approaching the suspended animal from the dorsal position, a 1 mLsyringe fitted with a gavage needle was inserted orally into the mouseand descended into the trachea just above the carina. The dose wasinstilled into the lungs and the gavage needle was then immediatelyremoved. Exenatide was administered by subcutaneous (SC) injection usinga 20 G1 inch 1 mL tuberculin syringe. Blood samples of approximately0.07 mL were collected from the tail vein at each time point. Thesesamples were tested for concentrations of glucose. The glucosemeasurement was made in duplicate with Glucometer Elite (Bayer Corp.,Elkart, Ind.) glucose monitors. The upper limit of quantitation (ULOQ)of these glucometers is 600 mg/dl. This value (600 mg/dl) was used forcalculation of descriptive statistics when the ULOQ was exceeded. Groupmean blood glucose concentrations (mg/dl) are given in FIG. 17.Statistical evaluation was made with a 2-tailed t-test at a P value of0.05 and compared selected time points to T=0 value for that treatmentgroup.

TABLE 6 Study Design Total Dose of Route of Number of Test No. of GroupControl/Test Adminis- Animals/ Article Dosing No. Article tration Gender(μg/mouse) Days 1 mPEGSPC_(5k)- IT 9 M 1000 1 N^(ter)-GLP-1 2 Vehicle IT6 M NA 1 3 Exenatide SC 3 M 0.1 1

The objective of this study, to demonstrate pharmacological activity ofthe PEGylated and releasable GLP-1 conjugate,mPEGSPC_(5k)-N^(ter)-GLP-1, when administered intratracheally (IT) inthe mouse, was achieved.

Mice were administered mPEGSPC_(5k)-N^(ter)-GLP-1 or vehicleintratracheally and were tested for blood glucose and plasma insulinconcentrations at various time points post administration. Exenatideadministered SC was used as a positive control.

A significant reduction in blood glucose was observed at the 1 and 3hour time points in the group administered exenatide. Blood glucose hadreturned to baseline by the next time point (24 hrs).

mPEGSPC_(5k)-N^(ter)-GLP-1 caused an overall reduction in blood glucoseprofile from the 2 hour time point to the 31 hour (last) time point whencompared to the vehicle (FIG. 17). When group means at individual timepoints were compared to pre-dose values, glucose suppression at 2 and 4hours reached statistical significance. This was true whether the −24 or0 hour pre-dose values were used for the comparison. Glucose reductionat these time points did not reach significance when compared to thevalues for the vehicle group at the corresponding time point. It shouldbe noted that there were only 3 mice per non pre-dose time point andthat there is considerable variation between individual mouse values.

Intact mPEGSPC_(5k)-N^(ter)-GLP-1 is assumed not to have pharmacologicalactivity. This is presumed because of the interference of the N-terminalPEG with the interaction of the GLP-1 ligand to its receptor. Thereleased GLP-1 moiety is expected to have a low level of pharmacologicalactivity when compared to native GLP-1 due to a residual carbonyl moietyat the N-terminus (which would likely also interfere withreceptor-ligand binding). This lower intrinsic activity of the cleavageproduct may have contributed to the less pronounced pharmacologicaleffect of the mPEGSPC_(5k)-N^(ter)-GLP-1 relative to that ofPEG2-CAC-Fmoc_(4k)-N^(ter)-GLP-1. The observation of in vivopharmacological activity and the occurrence of the activity maximum 4hours after administration are consistent with in vivo cleavage ofmPEGSPC_(5k)-N^(ter)-GLP-1 and release of a form of active GLP-1.

Though it does not reach statistical significance at individual timepoints, IT administration of mPEGSPC_(5k)-N^(ter)-GLP-1 results in anapparent continued glucose suppression after the 4 hour time pointsrelative to the vehicle group.

No adverse effect attributed to compound was observed in these studies.

In sum, the results of this experiment, indicate that the intratrachealadministration of mPEGSPC_(5k)-N^(ter)-GLP-1, a cleavable GLP-1conjugate, resulted in a statistically significant suppression of bloodglucose in a male diabetic mouse (BKS.Cg-+Lepr db/+Lepr db/01aHsd)model.

The foregoing embodiments and advantages are merely exemplary and arenot to be construed as limiting the present invention. The descriptionof the present invention is intended to be illustrative, and not tolimit the scope of the claims. Many alternatives, modifications, andvariations will be apparent to those skilled in the art.

Having now fully described this invention, it will be understood tothose of ordinary skill in the art that the methods of the presentinvention can be carried out with a wide and equivalent range ofconditions, formulations, and other parameters without departing fromthe scope of the invention or any embodiments thereof.

All patents and publications cited herein are hereby fully incorporatedby reference in their entirety. The citation of any publication is forits disclosure prior to the filing date and should not be construed asan admission that such publication is prior art or that the presentinvention is not entitled to antedate such publication by virtue ofprior invention.

What is claimed is:
 1. A GLP-1 polymer conjugate comprising a GLP-1moiety releasably attached at an amine to a water-soluble polymer,wherein the conjugate is formed by contacting the GLP-1 moiety with apolymeric reagent of the following structure:

where: POLY¹ is a first water-soluble polymer; POLY² is a secondwater-soluble polymer; X¹ is a first spacer moiety; X² is a secondspacer moiety; H_(α) is an ionizable hydrogen atom; R¹ is H or anorganic radical; R² is H or an organic radical; and (FG) is a functionalgroup capable of reacting with an amino group of the GLP-1 moiety. 2.The GLP-1 polymer conjugate of claim 1, wherein each of the firstwater-soluble polymer and second water-soluble polymer is independentlyselected from poly(alkylene glycol), poly(oxyethylated polyol),poly(olefinic alcohol), poly(vinylpyrrolidone),poly(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate),poly(saccharide), poly(α-hydroxy acid), poly(vinyl alcohol),polyphosphazene, polyoxazoline, poly(N-acryloylmorpholine), andcopolymers and terpolymers thereof.
 3. The GLP-1 polymer conjugate ofclaim 2, wherein each of the first water-soluble polymer and secondwater-soluble polymer is a polyethylene glycol.
 4. The GLP-1 polymerconjugate of claim 1, wherein the releasably attached GLP-1 moiety isreleasably attached via a linker selected from carbamate, carboxylateester, phosphate ester, anhydride, acetal, ketal, acyloxyalkyl ether,imine, orthoester, thioester, thiolester, and carbonate.
 5. The GLP-1polymer conjugate of claim 1, wherein the releasably attached GLP-1moiety is releasably attached via a linker selected from carbamate,carboxylate ester, and carbonate.
 6. The GLP-1 polymer conjugate ofclaim 1, wherein each of the first water-soluble polymer and secondwater-soluble polymer has a molecular weight ranging from about 500daltons to about 80,000 daltons.
 7. The GLP-1 polymer conjugate of claim6, wherein each of the first water-soluble polymer and secondwater-soluble polymer has a molecular weight ranging from about 1000daltons to about 40,000 daltons.
 8. The GLP-1 polymer conjugate of claim1, wherein the GLP-1 moiety is glycosylated.
 9. The GLP-1 polymerconjugate of claim 1, wherein the GLP-1 moiety possesses an N-methylsubstituent.